Influence of TGFBR2, TGFB3, DNMT1, and DNMT3A Knockdowns on CTGF, TGFBR2, and DNMT3A in Neonatal and Adult Human Dermal Fibroblasts Cell Lines.

Dermal fibroblasts are responsible for the production of the extracellular matrix that undergoes significant changes during the skin aging process. These changes are partially controlled by the TGF-ß signaling, which regulates tissue homeostasis dependently on several genes, including CTGF and DNA methyltransferases. To investigate the potential differences in the regulation of the TGF-ß signaling and related molecular pathways at distinct developmental stages, we silenced the expression of TGFB1, TGFB3, TGFBR2, CTGF, DNMT1, and DNMT3A in the neonatal (HDF-N) and adult (HDF-A) human dermal fibroblasts using the RNAi method. Through Western blot, we analyzed the effects of the knockdowns of these genes on the level of the CTGF, TGFBR2, and DNMT3A proteins in both cell lines. In the in vitro assays, we observed that CTGF level was decreased after knockdown of DNMT1 in HDF-N but not in HDF-A. Similarly, the level of DNMT3A was decreased only in HDF-N after silencing of TGFBR2, TGFB3, or DNMT1. TGFBR2 level was lower in HDF-N after knockdown of TGFB3, DNMT1, or DNMT3A, but it was higher in HDF-A after TGFB1 silencing. The reduction of TGFBR2 after silencing of DNMT3A and vice versa in neonatal cells only suggests the developmental stage-specific interactions between these two genes. However, additional studies are needed to explain the dependencies between analyzed proteins.

Dual Role of the Histone Variant H2A.Z in Transcriptional Regulation of Stress-Response Genes.

The influence of the histone variant H2A.Z on transcription remains a long-standing conundrum. Here, by analyzing the actin-related protein6 mutant, which is impaired in H2A.Z deposition, and by H2A.Z profiling in stress conditions, we investigated the impact of this histone variant on gene expression in Arabidopsis thaliana We demonstrate that the arp6 mutant exhibits anomalies in response to osmotic stress. Indeed, stress-responsive genes are overrepresented among those hyperactive in arp6. In wild-type plants, these genes exhibit high levels of H2A.Z in the gene body. Furthermore, we observed that in drought-responsive genes, levels of H2A.Z in the gene body correlate with transcript levels. H2A.Z occupancy, but not distribution, changes in parallel with transcriptional changes. In particular, we observed H2A.Z loss upon transcriptional activation and H2A.Z gain upon repression. These data suggest that H2A.Z has a repressive role in transcription and counteracts unwanted expression in noninductive conditions. However, reduced activity of some genes in arp6 is associated with distinct behavior of H2A.Z at their +1 nucleosome, which exemplifies the requirement of this histone for transcription. Our data support a model where H2A.Z in gene bodies has a strong repressive effect on transcription, whereas in +1 nucleosomes, it is important for maintaining the activity of some genes.

Mechanistic determinants of MBNL activity.

Muscleblind-like (MBNL) proteins are critical RNA processing factors in development. MBNL activity is disrupted in the neuromuscular disease myotonic dystrophy type 1 (DM1), due to the instability of a non-coding microsatellite in the DMPK gene and the expression of CUG expansion (CUGexp) RNAs. Pathogenic interactions between MBNL and CUGexp RNA lead to the formation of nuclear complexes termed foci and prevent MBNL function in pre-mRNA processing. The existence of multiple MBNL genes, as well as multiple protein isoforms, raises the question of whether different MBNL proteins possess unique or redundant functions. To address this question, we coexpressed three MBNL paralogs in cells at equivalent levels and characterized both specific and redundant roles of these proteins in alternative splicing and RNA foci dynamics. When coexpressed in the same cells, MBNL1, MBNL2 and MBNL3 bind the same RNA motifs with different affinities. While MBNL1 demonstrated the highest splicing activity, MBNL3 showed the lowest. When forming RNA foci, MBNL1 is the most mobile paralog, while MBNL3 is rather static and the most densely packed on CUGexp RNA. Therefore, our results demonstrate that MBNL paralogs and gene-specific isoforms possess inherent functional differences, an outcome that could be enlisted to improve therapeutic strategies for DM1.

Inter-population Differences in Retrogene Loss and Expression in Humans.

Gene retroposition leads to considerable genetic variation between individuals. Recent studies revealed the presence of at least 208 retroduplication variations (RDVs), a class of polymorphisms, in which a retrocopy is present or absent from individual genomes. Most of these RDVs resulted from recent retroduplications. In this study, we used the results of Phase 1 from the 1000 Genomes Project to investigate the variation in loss of ancestral (i.e. shared with other primates) retrocopies among different human populations. In addition, we examined retrocopy expression levels using RNA-Seq data derived from the Ilumina BodyMap project, as well as data from lymphoblastoid cell lines provided by the Geuvadis Consortium. We also developed a new approach to detect novel retrocopies absent from the reference human genome. We experimentally confirmed the existence of the detected retrocopies and determined their presence or absence in the human genomes of 17 different populations. Altogether, we were able to detect 193 RDVs; the majority resulted from retrocopy deletion. Most of these RDVs had not been previously reported. We experimentally confirmed the expression of 11 ancestral retrogenes that underwent deletion in certain individuals. The frequency of their deletion, with the exception of one retrogene, is very low. The expression, conservation and low rate of deletion of the remaining 10 retrocopies may suggest some functionality. Aside from the presence or absence of expressed retrocopies, we also searched for differences in retrocopy expression levels between populations, finding 9 retrogenes that undergo statistically significant differential expression.

RetrogeneDB--a database of animal retrogenes.

Retrocopies of protein-coding genes, reverse transcribed and inserted into the genome copies of mature RNA, have commonly been categorized as pseudogenes with no biological importance. However, recent studies showed that they play important role in the genomes evolution and shaping interspecies differences. Here, we present RetrogeneDB, a database of retrocopies in 62 animal genomes. RetrogeneDB contains information about retrocopies, their genomic localization, parental genes, ORF conservation, and expression. To our best knowledge, this is the most complete retrocopies database providing information for dozens of species previously never analyzed in the context of protein-coding genes retroposition. The database is available at http://retrogenedb.amu.edu.pl.

DNA methylation dysregulation patterns in the 1p36 region instability.

In the monosomy 1p36 deletion syndrome, the role of DNA methylation in the genomic stability of the 1p36 region remains elusive. We hypothesize that changes in the methylation pattern at the 1p36 breakpoint hotspot region influenced the chromosomal breakage leading to terminal deletions. From the monosomy 1p36 material collection, four cases with 4.0 to 5.5 Mb terminal deletions and their parents were investigated. DNA samples were assessed by targeted bisulfite sequencing (NimbleGen SeqCap Epi) to examine DNA methylation status in the 1p36 hotspot region at single-base resolution as compared to the chromosomal hotspot regions, 9p22, 18q21.1, and 22q11.2. Additionally, in in silico assessment, the mean GC content of various classes of repeats in the genome and especially in the breakpoint regions was evaluated. A complex landscape of DNA methylation in the 1p36 breakpoint hotspot region was found. Changes in DNA methylation level in the vicinity of the breakpoint in the child's DNA when compared to parents' and control DNA were observed, with a shift from 15.1 to 70.8% spanning the breakpoint region. In the main classes of evaluated repeats, higher mean GC contents in the 1p36 breakpoint region (47.06%), 22q11.2 (48.47%), and 18q21.1 (44.21%) were found, compared to the rest of the genome (40.78%). The 9p22 region showed a lower GC content (39.42%) compared to the rest of the genome. Both dysregulation of DNA methylation and high GC content were found to be specific for the 1p36 breakpoint hotspot region suggesting that methylation abnormalities could contribute to aberrations at 1p36.

Accurate long-read transcript discovery and quantification at single-cell, pseudo-bulk and bulk resolution with Isosceles.

Accurate detection and quantification of mRNA isoforms from nanopore long-read sequencing remains challenged by technical noise, particularly in single cells. To address this, we introduce Isosceles, a computational toolkit that outperforms other methods in isoform detection sensitivity and quantification accuracy across single-cell, pseudo-bulk and bulk resolution levels, as demonstrated using synthetic and biologically-derived datasets. Here we show Isosceles improves the fidelity of single-cell transcriptome quantification at the isoform-level, and enables flexible downstream analysis. As a case study, we apply Isosceles, uncovering coordinated splicing within and between neuronal differentiation lineages. Isosceles is suitable to be applied in diverse biological systems, facilitating studies of cellular heterogeneity across biomedical research applications.

CRISPR activation screens identify the SWI/SNF ATPases as suppressors of ferroptosis.

Ferroptosis is an iron-dependent cell death mechanism characterized by the accumulation of toxic lipid peroxides and cell membrane rupture. GPX4 (glutathione peroxidase 4) prevents ferroptosis by reducing these lipid peroxides into lipid alcohols. Ferroptosis induction by GPX4 inhibition has emerged as a vulnerability of cancer cells, highlighting the need to identify ferroptosis regulators that may be exploited therapeutically. Through genome-wide CRISPR activation screens, we identify the SWI/SNF (switch/sucrose non-fermentable) ATPases BRM (SMARCA2) and BRG1 (SMARCA4) as ferroptosis suppressors. Mechanistically, they bind to and increase chromatin accessibility at NRF2 target loci, thus boosting NRF2 transcriptional output to counter lipid peroxidation and confer resistance to GPX4 inhibition. We further demonstrate that the BRM/BRG1 ferroptosis connection can be leveraged to enhance the paralog dependency of BRG1 mutant cancer cells on BRM. Our data reveal ferroptosis induction as a potential avenue for broadening the efficacy of BRM degraders/inhibitors and define a specific genetic context for exploiting GPX4 dependency.

ERISdb: a database of plant splice sites and splicing signals.

Splicing is one of the major contributors to observed spatiotemporal diversification of transcripts and proteins in metazoans. There are numerous factors that affect the process, but splice sites themselves along with the adjacent splicing signals are critical here. Unfortunately, there is still little known about splicing in plants and, consequently, further research in some fields of plant molecular biology will encounter difficulties. Keeping this in mind, we performed a large-scale analysis of splice sites in eight plant species, using novel algorithms and tools developed by us. The analyses included identification of orthologous splice sites, polypyrimidine tracts and branch sites. Additionally we identified putative intronic and exonic cis-regulatory motifs, U12 introns as well as splice sites in 45 microRNA genes in five plant species. We also provide experimental evidence for plant splice sites in the form of expressed sequence tag and RNA-Seq data. All the data are stored in a novel database called ERISdb and are freely available at http://lemur.amu.edu.pl/share/ERISdb/.

Sequence variants contributing to dysregulated inflammatory responses across keratoconic cone surface in adolescent patients with keratoconus.

Background: Keratoconus (KTCN) is the most common corneal ectasia resulting in a conical shape of the cornea. Here, genomic variation in the corneal epithelium (CE) across the keratoconic cone surface in patients with KTCN and its relevance in the functioning of the immune system were assessed. Methods: Samples from four unrelated adolescent patients with KTCN and two control individuals were obtained during the CXL and PRK procedures, respectively. Three topographic regions, central, middle, and peripheral, were separated towards the whole-genome sequencing (WGS) study embracing a total of 18 experimental samples. The coding and non-coding sequence variation, including structural variation, was assessed and then evaluated together with the previously reported transcriptomic outcomes for the same CE samples and full-thickness corneas. Results: First, pathway enrichment analysis of genes with identified coding variants pointed to "Antigen presentation" and "Interferon alpha/beta signaling" as the most overrepresented pathways, indicating the involvement of inflammatory responses in KTCN. Both coding and non-coding sequence variants were found in genes (or in their close proximity) linked to the previously revealed KTCN-specific cellular components, namely, "Actin cytoskeleton", "Extracellular matrix", "Collagen-containing extracellular matrix", "Focal adhesion", "Hippo signaling pathway", and "Wnt signaling" pathways. No genomic heterogeneity across the corneal surface was found comparing the assessed topographic regions. Thirty-five chromosomal regions enriched in both coding and non-coding KTCN-specific sequence variants were revealed, with a most representative 5q locus previously recognized as involved in KTCN. Conclusion: The identified genomic features indicate the involvement of innate and adaptive immune system responses in KTCN pathogenesis.

The Impaired Wound Healing Process Is a Major Factor in Remodeling of the Corneal Epithelium in Adult and Adolescent Patients With Keratoconus.

Purpose: Keratoconus (KTCN) is the most common corneal ectasia, characterized by pathological cone formation. Here, to provide an insight into the remodeling of the corneal epithelium (CE) during the course of the disease, we evaluated topographic regions of the CE of adult and adolescent patients with KTCN. Methods: The CE samples from 17 adult and 6 adolescent patients with KTCN, and 5 control CE samples were obtained during the CXL and PRK procedures, respectively. Three topographic regions, central, middle, and peripheral, were separated toward RNA sequencing and MALDI-TOF/TOF Tandem Mass Spectrometry. Data from transcriptomic and proteomic investigations were consolidated with the morphological and clinical findings. Results: The critical elements of the wound healing process, epithelial-mesenchymal transition, cell-cell communications, and cell-extracellular matrix interactions were altered in the particular corneal topographic regions. Abnormalities in pathways of neutrophils degranulation, extracellular matrix processing, apical junctions, IL, and IFN signaling were revealed to cooperatively disorganize the epithelial healing. Deregulation of the epithelial healing, G2M checkpoints, apoptosis, and DNA repair pathways in the middle CE topographic region in KTCN explains the presence of morphological changes in the corresponding doughnut pattern (a thin cone center surrounded by a thickened annulus). Despite similar morphological characteristics of CE samples in adolescents and adults with KTCN, their transcriptomic features were different. Values of the posterior corneal elevation differentiated adults with KTCN from adolescents with KTCN and correlated with the expression of TCHP, SPATA13, CNOT3, WNK1, TGFB2, and KRT12 genes. Conclusions: Identified molecular, morphological, and clinical features indicate the effect of impaired wound healing on corneal remodeling in KTCN CE.

An allosteric pan-TEAD inhibitor blocks oncogenic YAP/TAZ signaling and overcomes KRAS G12C inhibitor resistance.

The Hippo pathway is a key growth control pathway that is conserved across species. The downstream effectors of the Hippo pathway, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), are frequently activated in cancers to drive proliferation and survival. Based on the premise that sustained interactions between YAP/TAZ and TEADs (transcriptional enhanced associate domain) are central to their transcriptional activities, we discovered a potent small-molecule inhibitor (SMI), GNE-7883, that allosterically blocks the interactions between YAP/TAZ and all human TEAD paralogs through binding to the TEAD lipid pocket. GNE-7883 effectively reduces chromatin accessibility specifically at TEAD motifs, suppresses cell proliferation in a variety of cell line models and achieves strong antitumor efficacy in vivo. Furthermore, we uncovered that GNE-7883 effectively overcomes both intrinsic and acquired resistance to KRAS (Kirsten rat sarcoma viral oncogene homolog) G12C inhibitors in diverse preclinical models through the inhibition of YAP/TAZ activation. Taken together, this work demonstrates the activities of TEAD SMIs in YAP/TAZ-dependent cancers and highlights their potential broad applications in precision oncology and therapy resistance.

NuA4 and H2A.Z control environmental responses and autotrophic growth in Arabidopsis.

Nucleosomal acetyltransferase of H4 (NuA4) is an essential transcriptional coactivator in eukaryotes, but remains poorly characterized in plants. Here, we describe Arabidopsis homologs of the NuA4 scaffold proteins Enhancer of Polycomb-Like 1 (AtEPL1) and Esa1-Associated Factor 1 (AtEAF1). Loss of AtEAF1 results in inhibition of growth and chloroplast development. These effects are stronger in the Atepl1 mutant and are further enhanced by loss of Golden2-Like (GLK) transcription factors, suggesting that NuA4 activates nuclear plastid genes alongside GLK. We demonstrate that AtEPL1 is necessary for nucleosomal acetylation of histones H4 and H2A.Z by NuA4 in vitro. These chromatin marks are diminished genome-wide in Atepl1, while another active chromatin mark, H3K9 acetylation (H3K9ac), is locally enhanced. Expression of many chloroplast-related genes depends on NuA4, as they are downregulated with loss of H4ac and H2A.Zac. Finally, we demonstrate that NuA4 promotes H2A.Z deposition and by doing so prevents spurious activation of stress response genes.

PacBio-Based Protocol for Bacterial Genome Assembly.

Acquisition of high-quality bacterial genomes is fundamental, while having in mind investigation of subtitle intraspecies variation in addition to development of sensitive species-specific tools for detection and identification of the pathogens. In this view, Pacific Biosciences technology seems highly tempting taking into consideration over 10,000 bp length of the generated reads. In this work, we describe a bacterial genome assembly pipeline based on open-source software that might be handled also by non-bioinformaticians interested in transformation of sequencing data into reliable biological information. With the use of this method, we successfully closed six Dickeya solani genomes, while the assembly process was run just on a slightly improved desktop computer.

Characterization of Ocular Surface Microbial Profiles Revealed Discrepancies between Conjunctival and Corneal Microbiota.

The ocular microbiome composition has only been partially characterized. Here, we used RNA-sequencing (RNA-Seq) data to assess microbial diversity in human corneal tissue. Additionally, conjunctival swab samples were examined to characterize ocular surface microbiota. Short RNA-Seq reads, obtained from a previous transcriptome study of 50 corneal tissues, were mapped to the human reference genome GRCh38 to remove sequences of human origin. The unmapped reads were then used for taxonomic classification by comparing them with known bacterial, archaeal, and viral sequences from public databases. The components of microbial communities were identified and characterized using both conventional microbiology and polymerase chain reaction (PCR) techniques in 36 conjunctival swabs. The majority of ocular samples examined by conventional and molecular techniques showed very similar microbial taxonomic profiles, with most of the microorganisms being classified into Proteobacteria, Firmicutes, and Actinobacteria phyla. Only 50% of conjunctival samples exhibited bacterial growth. The PCR detection provided a broader overview of positive results for conjunctival materials. The RNA-Seq assessment revealed significant variability of the corneal microbial communities, including fastidious bacteria and viruses. The use of the combined techniques allowed for a comprehensive characterization of the eye microbiome's elements, especially in aspects of microbiota diversity.

Further evaluation of differential expression of keratoconus candidate genes in human corneas.

BACKGROUND: Keratoconus (KTCN) is a progressive eye disease, characterized by changes in the shape and thickness of the cornea that results in loss of visual acuity. While numerous KTCN candidate genes have been identified, the genetic etiology of the disease remains undetermined. To further investigate and verify the contribution of particular genetic factors to KTCN, we assessed 45 candidate genes previously indicated as involved in KTCN etiology based on transcriptomic and genomic data. METHODS: The RealTime ready Custom Panel, covering 45 KTCN candidate genes and two reference transcripts, has been designed. Then, the expression profiles have been assessed using the RT-qPCR assay in six KTCN and six non-KTCN human corneas, obtained from individuals undergoing a penetrating keratoplasty procedure. RESULTS: In total, 35 genes exhibiting differential expression between KTCN and non-KTCN corneas have been identified. Among these genes were ones linked to the extracellular matrix formation, including collagen synthesis or the TGF-ß, Hippo, and Wnt signaling pathways. The most downregulated transcripts in KTCN corneas were CTGF, TGFB3, ZNF469, COL5A2, SMAD7, and SPARC, while TGFBI and SLC4A11 were the most upregulated ones. Hierarchical clustering of expression profiles demonstrated almost clear separation between KTCN and non-KTCN corneas. The gene expression levels determined using RT-qPCR showed a strong correlation with previous RNA sequencing (RNA-Seq) results. CONCLUSIONS: A strong correlation between RT-qPCR and earlier RNA-Seq data confirms the possible involvement of genes from collagen synthesis and the TGF-ß, Hippo, and Wnt signaling pathways in KTCN etiology. Our data also revealed altered expression of several genes, such as LOX, SPARC, and ZNF469, in which single nucleotide variants have been frequently identified in KTCN. These findings further highlight the heterogeneous nature of KTCN.

Multiple Differentially Methylated Regions Specific to Keratoconus Explain Known Keratoconus Linkage Loci.

Purpose: Keratoconus (KTCN) is a complex eye disorder resulting in loss of visual function. Its development is affected by genetic and environmental components. The aim of this study was to unravel the role of epigenetic factors in KTCN. Methods: To verify if DNA methylation may play a role in KTCN development, reduced representation bisulfite sequencing of five KTCN and five non-KTCN human corneas was performed. Results: Multiple KTCN-specific differentially methylated regions were detected and many of them overlap previously identified KTCN linkage loci (3p14.3, 5q35.2, 13q32.3, 15q24.1, and 20p13) and chromosome arms that have been linked to KTCN (2q, 4q, 5p, 9p, 14q, and 17q). Reanalysis of the previously described RNA sequencing dataset of 25 KTCN and 25 non-KTCN human corneas revealed that 12 genes downregulated in KTCN and 6 upregulated genes overlapped or were located in the near vicinity of the identified differentially methylated regions. Particularly interesting were the DNA methylation changes in WNT3 and WNT5A encoding Wnt ligands, as they provide a potential explanation for the Wnt signaling pathway dysregulation observed in KTCN. Conclusions: We presented the results of data analysis from the first study of DNA methylation changes in human KTCN corneas compared to non-KTCN samples. We were able to identify genomic regions with distinct patterns of DNA hypo- and hypermethylation and link them to previously found KTCN susceptibility loci as well as transcriptomic disruption of Wnt signaling pathway observed in KTCN.

Comparison of Highly and Weakly Virulent Dickeya solani Strains, With a View on the Pangenome and Panregulon of This Species.

Bacteria belonging to the genera Dickeya and Pectobacterium are responsible for significant economic losses in a wide variety of crops and ornamentals. During last years, increasing losses in potato production have been attributed to the appearance of Dickeya solani. The D. solani strains investigated so far share genetic homogeneity, although different virulence levels were observed among strains of various origins. The purpose of this study was to investigate the genetic traits possibly related to the diverse virulence levels by means of comparative genomics. First, we developed a new genome assembly pipeline which allowed us to complete the D. solani genomes. Four de novo sequenced and ten publicly available genomes were used to identify the structure of the D. solani pangenome, in which 74.8 and 25.2% of genes were grouped into the core and dispensable genome, respectively. For D. solani panregulon analysis, we performed a binding site prediction for four transcription factors, namely CRP, KdgR, PecS and Fur, to detect the regulons of these virulence regulators. Most of the D. solani potential virulence factors were predicted to belong to the accessory regulons of CRP, KdgR, and PecS. Thus, some differences in gene expression could exist between D. solani strains. The comparison between a highly and a low virulent strain, IFB0099 and IFB0223, respectively, disclosed only small differences between their genomes but significant differences in the production of virulence factors like pectinases, cellulases and proteases, and in their mobility. The D. solani strains also diverge in the number and size of prophages present in their genomes. Another relevant difference is the disruption of the adhesin gene fhaB2 in the highly virulent strain. Strain IFB0223, which has a complete adhesin gene, is less mobile and less aggressive than IFB0099. This suggests that in this case, mobility rather than adherence is needed in order to trigger disease symptoms. This study highlights the utility of comparative genomics in predicting D. solani traits involved in the aggressiveness of this emerging plant pathogen.

Hypermethylation of TRIM59 and KLF14 Influences Cell Death Signaling in Familial Alzheimer's Disease.

Epigenetic mechanisms play an important role in the development and progression of various neurodegenerative diseases. Abnormal methylation of numerous genes responsible for regulation of transcription, DNA replication, and apoptosis has been linked to Alzheimer's disease (AD) pathology. We have recently performed whole transcriptome profiling of familial early-onset Alzheimer's disease (fEOAD) patient-derived fibroblasts. On this basis, we demonstrated a strong dysregulation of cell cycle checkpoints and DNA damage response (DDR) in both fibroblasts and reprogrammed neurons. Here, we show that the aging-correlated hypermethylation of KLF14 and TRIM59 genes associates with abnormalities in DNA repair and cell cycle control in fEOAD. Based on the resulting transcriptome networks, we found that the hypermethylation of KLF14 might be associated with epigenetic regulation of the chromatin organization and mRNA processing followed by hypermethylation of TRIM59 likely associated with the G2/M cell cycle phase and p53 role in DNA repair with BRCA1 protein as the key player. We propose that the hypermethylation of KLF14 could constitute a superior epigenetic mechanism for TRIM59 hypermethylation. The methylation status of both genes affects genome stability and might contribute to proapoptotic signaling in AD. Since this study combines data obtained from various tissues from AD patients, it reinforces the view that the genetic methylation status in the blood may be a valuable predictor of molecular processes occurring in affected tissues. Further research is necessary to define a detailed role of TRIM59 and KLF4 in neurodegeneration of neurons.

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.