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

Abstract

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-β. 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-β pathology and promote cell cycle re-entry-driven cell death of postmitotic neurons in AD.

Keywords

Alzheimer’s disease BRCA1 DNA damage response cell cycle re-entry presenilin 1 ubiquitination

INTRODUCTION

Alzheimer’s disease (AD) is characterized by massive neuronal death leading to progressive loss of cognitive function. Familial early-onset AD (fEOAD) is in majority caused by mutations in PSEN1 encoding presenilin 1 (PS1), while late-onset AD (LOAD) is linked with mutations in PSEN2 encoding presenilin 2 (PS2) or APP encoding amyloid-β precursor protein (AβPP). Presenilins are major components of the catalytic core of the γ-secretase complex reprocessing amyloid-β (Aβ). Regardless of genetic status, the Aβ toxicity is present in both fEOAD and LOAD or sporadic AD [1]. Unfortunately, the pathomechanisms associated with PS1 and Aβ processing at the prodromal stage of AD remain still not fully understood.

DNA damage response (DDR) has been implicated in neuronal death of various neurodegenerative diseases, including AD [2, 3]. DDR includes activation of the ATM (Ataxia Telangiectasia Mutated) and ATR (Ataxia Telangiectasia and Rad3 Related) signaling pathway, in which the BRCA1 (breast cancer 1) protein is one of the key players [4]. BRCA1 maintains genomic stability by regulation of cell cycle checkpoints (CCC), DNA repair, apoptosis, and protein ubiquitination due to its E3 ubiquitin ligase activity [5]. Interestingly, BRCA1 is needed for apoptosis of neural precursors during their asymmetrical divisions, while in mature neurons its activity must be extinguished [6]. In addition, many of the proteins involved in CCC and DDR have been found in neurons, but their neuronal functions are still not fully understood [7]. Recently, a depletion of non-phosphorylated BRCA1 have been associated with accumulation of Aβ in AD patients’ postmortem brains [8]. These findings paved the way for further research on the role of BRCA1 in degenerating brain.

To elucidate the significance of the BRCA1 protein in AD, we used different types of fEOAD patients-derived cells, including primary dermal fibroblast and neuronal cells (neurons and neural stem cells) differentiated from induced pluripotent stem cell (iPSC) reprogrammed from the above-mentioned fibroblasts. We have started our research by next generation sequencing of total RNA from fEOAD patients-derived fibroblasts, which revealed a wide spectrum of changes in BRCA1 functions, both related to DDR and to CCC in fEOAD patients. On this basis, using different types of cells derived from fEOAD patients, we studied activation status and cellular localization of the BRCA1 protein, which we consider crucial in the earliest stages of cell death in AD. This study provides the first data on molecular mechanisms linking BRCA1 to AD, including on one hand a proteasome-dependent PS1 degradation and a dysregulation of cell cycle checkpoints with DDR induction on the other.

MATERIALS AND METHODS

Patients and control subjects

Ethical issues

The Ethics Committee of the Department of Neurology of the Central Clinical Hospital of the Ministry of Interior Affairs and Administration in Warsaw approved the protocol of the acquisition of skin biopsies (Decision no. 31/2013). Written informed consent for participation in this research and for publication was obtained from patients (or they legal representatives) and controls, according to the Declaration of Helsinki (BMJ 1991; 302:1194).

Genetic screening

Patients and healthy controls were screened by Sanger sequencing. Genomic DNA was extracted from peripheral blood leukocytes using salting-out method. Genomic DNA was sequenced for exons 3–13 of PSEN1, exons 1–12 of PSEN2, and exons 16 and 17 of APP, as we described previously [9, 10].

Diagnostics

In this study, we used primary fibroblast cell lines derived from fEOAD patients with mutations in PSEN1 and from healthy, age- and sex-matched donors. Summary of patients’ genetic status is provided in Supplementary Table 1. Neuropsychological and clinical description of the patients is provided in Supplementary Method 1. Healthy donors were screened for: 1) genetic status, confirming no PSEN1, PSEN2, APP mutations, 2) cognitive condition, confirming a normal score of the Mini-Mental State Examination (MMSE) [11], 3) familial history, confirming no dementia or relative neurological disorders present in the family across at least 3 generations, and 4) excluded for strokes and other neurological diseases (Supplementary Table 2).

Skin biopsies and primary cell culture of fibroblasts

The skin biopsies of 4-mm punch collected from the upper arm were devoid of fat tissue, blood vessels and epidermis and incubated at 37°C, 5% CO₂ for 1 h in 2 mg/ml collagenase (Sigma-Aldrich) and 0.1 mg/ml DNase I (Sigma-Aldrich) in DMEM supplemented with 4.5 g/l glucose, 4 mM L-glutamine, 5 mM sodium pyruvate, 10,000 U/ml penicillin, and 10 mg/ml streptomycin (Thermo Fisher Scientific, Gibco). Fibroblasts migrated out of the biopsies within the first week and were cultured in DMEM with 10% fetal bovine serum (Thermo Fisher Scientific, Gibco) for maximum 10 passages. The cell lines were mycoplasma-negative as tested by PCR Mycoplasma kit (MP Biomedicals).

Reprogramming of fibroblasts into iPSC

iPSC were generated by reprogramming 5×10⁵ primary fibroblasts with Sendai virus co-expressing Oct4, Sox2, Klf4, and cMyc at multiplicity of infection (MOI) = 2 as we reported before [12]. The resultant iPSC were validated for the presence of pluripotency markers and cultured in E8 medium (Thermo Fisher Scientific, Gibco) and Laminin-521-coated plates (BioLamina).

Neural induction of iPSC into NES cells and neuronal differentiation

Neuroepithelial-like stem (NES) cells were derived by neural induction of iPSC as we described previously [13]. Briefly, iPSC were induced in KOSR and N2B27 medium (Thermo Fisher Scientific, Gibco) with hNoggin (PreproTech), SB431542 (Sigma Aldrich), and CHIR99021 (StemMolecule). NES cells were cultured at 37°C in 5% CO₂ in poly-ornithine (Sigma-Aldrich) and laminin 2020 (Sigma-Aldrich) coating in DMEM/F12 with Glutamax, N-2 supplement, B27 supplement, antibiotics, 10 ng/ml bFGF (Thermo Fisher Scientific, Gibco), and 10 ng/ml EGF (PreproTech). NES cells were tested for continuous expandability in the presence of bFGF2 and EGF, stable neuronal differentiation competence and neuronal markers profile. NES cells were differentiated into neurons for 14 days by cell culture upon EGF and bFGF2 removal, in Neurobasal-B27 medium (Thermo Fisher Scientific, Gibco) with 50 ng/ml NGF (PreproTech). NES cells and neurons were imaged with integrated contrast module in Leica DM IL LED microscope (Leica Microsystems). Total length of neurites was measured by NeuroJ plugin in FijiJ software.

RNA isolation, cDNA library preparation, and RNA sequencing

Total RNA was isolated from fibroblasts using RNeasy Mini Kit (Qiagen). RNA integrity was assessed using RNA BR Assay Kit on Qubit 2.0 and RNA 6000 Pico Kit on Bioanalyzer 2100 (Agilent). 500 ng of RNA at integrity number >8 was converted to cDNA using TruSeq Stranded Total RNA with Ribo-Zero kit (Illumina). Libraries were assessed qualitatively on Bioanalyzer 2100 using High Sensitivity DNA Kit and quantitatively on CFX96 Real-Time PCR system (Bio-Rad) using KAPA Library Quantification Kit (Kapa Biosystems), and then sequenced 2×76 bp on HiSeq2500 Illumina platform. At least 20 million reads per sample were obtained with mean quality score (Q30) >94%. The sequencing data were demultiplexed, converted to FASTQ files and deposited at the Sequence Read Archive database as BioProject PRJNA382346 (https://www.ncbi.nlm.nih.gov/bioproject/382346).

Bioinformatic analysis

Raw reads in FASTQ files were trimmed by Trimmomatic [14], rRNA removed using Bowtie2 [15] and aligned to human genome GRCh38 release 76 (NCBI) using STAR splice junction mapper [16]. The samples were filtered from low mapping quality (>50) using Bamtools [17]. PCR duplicates were removed with Picard tool (http://broadinstitute.github.io/picard/) and the samples were sorted using Bamtools [17]. The mapped reads were counted using Subread [18]. The FPKM (Fragments Per Kilobase Of Exon Per Million Fragments Mapped) normalization method was applied to quantify the transcripts expression [19]. On average, 90% of reads were uniquely mapped by STAR software and mapped length was 155 bp, QC filtering passed 96%, rRNA fraction 14% and PCR duplicates 28% (Supplementary Table 3). Genes differentially expressed among fEOAD and controls were identified by DESeq2 in R-CRAN Environment [20, 21]. Ensembl Gene ID annotations were functionally assigned to gene ontology (GO) terms using the BioMart database [22]. Differentially expressed genes were tested for signaling pathways and GO terms enrichment using Pathview and Graphviz R packages [23], Generally Applicable Gene-set Enrichment for Pathway Analysis (GAGE) [24], implementing Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways collection [25]. Biological network activation was predicted with the Ingenuity Pathways Analysis (http://www.Ingenuity.com). The significant canonical pathways were filtered by IPA algorithms and -log(p-value) cutoff 1.3 was calculated by the Fisher exact test right-tailed. The IPA activation z-score≤2 predicted a decreased activity and z-score≥2 an increased activity.

RT-qPCR validation of RNA-sequencing hits and of pluripotency and neural markers

Expression of pluripotency markers OCT4, NANOG, KLF4, CMYC, and of SEV encoding Sendai virus protein was tested by reverse-transcription PCR (RT-PCR), as described before [13]. Gene expression of selected RNA-seq hits in fibroblasts or neural markers in NES cells and neurons was validated by quantitative RT-PCR (RT-qPCR). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) and 1.5 μg was transcribed to cDNA with Superscript II kit (Thermo Fisher Scientific, Invitrogen). Next, 3 μl of cDNA was used for SYBR Green qPCR (Thermo Fisher Scientific, Applied Biosystem). We tested expression of selected RNA-seq hits related to DDR and CCC and of multipotent neural stem cells markers SOX1, NES, KI67, MASH, PLAGL1, immature neurons and growth cone marker DCX, early neuronal markers MAPT, NFL, TUBB3, NCAM, glutamatergic markers VGLUT, GLUL, NMDAR1, GABAergic marker GABA, and cholinergic marker CHAT. All primers were designed using GeneScript Primer Design tool (Supplementary Table 4). RT-qPCR data were normalized to housekeeping control GAPDH according to the ΔΔCT method [26]. The qPCR data were analyzed by nonparametric paired Wilcoxon signed rank test at 95% or 99% confidence for comparison between experimental groups.

Protein cell lysates and immunoblotting

Total cell lysates, cytosolic and nuclear fractions were obtained from fibroblasts, NES cells and neurons grown at 100% confluence, treated with 2 μM doxorubicin (Santa-Cruz Biotechnology, sc-200923) for 0, 6, 16, 24, or 30 h, or with 50 or 100 nM bortezomib (Cell Signaling, #2204) for 24 h. Cells were lysed with RIPA (Radio Immuno Precipitation Assay) buffer supplemented with protease inhibitor cocktail (Roth), phosphatase inhibitor cocktail II (Sigma-Aldrich), and 20 mM NaF, and sonicated for 10 cycles for 0.5 s each at 60% power (Bandelin Sonoplus HD 2070). For cytosolic and nuclear fractions, cell lysates were incubated in hypotonic buffer containing 20 mM HEPES, pH 8.0, 0.05% NP-40, 1 mM EDTA, 1 mM ditiothreitol, and protease and phosphatase inhibitors. Cells were centrifuged at 1000×g for 5 min at 4°C and pellets containing nuclei were resuspended in ice-cold RIPA and sonicated as described above. For immunoblotting the lysates were boiled for 5 min at 95°C in Laemmli buffer, separated by SDS–PAGE on 6 to 12% acrylamide gels, and transferred to nitrocellulose membrane (Bio-Rad). The membrane was blocked in 5% non-fat dry milk in TBST (100 mM NaCl, 10 mM Tris-HCl pH 7.4, 0.05% Tween-20) for 1 h at room temperature (RT). Primary antibodies were diluted as required (Supplementary Table 5) in 5% nonfat dry milk in TBST and the membrane was incubated overnight at 4°C. Then the membrane was washed and probed with secondary antibodies (Bio-Rad) at the dilution of 1:20,000 for 2 h at RT. Next, membranes were incubated in chemiluminescent reagents (Bio-Rad). Blue-sensitive films (Primax) were used for detection. The film was analyzed by densitometry to GAPDH used for loading standardization.

Flow cytometry measurements of cell cycle and cell viability

Percentage of fibroblasts cultured with 2 μM doxorubicin for 0, 6, 16, 24, or 30 h at SubG1/apoptotic, G1, S, or G2/M phase was determined by propidium iodide staining. Cells washed twice with ice-cold PBS were suspended in 70% ice-cold ethanol. Then, 1×10⁵ cells were incubated with propidium iodide for 15 min at RT in the dark. Fluorescence was recorded with FACScan and CELLQuest software (Becton Dickinson).

Immunoprecipitation of PS1

Cells were lysed in the RIPA buffer. Protein G-agarose beads (Invitrogen, cat. no. 15920-010) were washed twice in PBS, blocked in 5% BSA in PBS for 2 h at 4°C and centrifuged at 14,000 x g for 1 min at 4°C. The extracts containing 0.5 mg of protein were pre-cleared with protein G-agarose beads, centrifuged at 14,000 x g for 1 min at 4°C and incubated for 2 h at 4°C with mouse monoclonal anti-PS1-FL antibody (Santa Cruz) (Supplementary Table 5). Aliquots of 40 μl protein G-agarose beads were added to the lysates and incubated o/n at 4°C. Then samples were centrifuged at 14,000 x g for 5 min at 4°C. Pellets and supernatants were boiled for 5 min at 95°C in Laemmli buffer, run SDS-PAGE and immunoblotted against BRCA1(Ser1524) (Supplementary Table 5).

Ubiquitination assay

Total cellular extracts immunoprecipitated with total BRCA1 (R&D Systems) or PS1-FL (Santa Cruz) as described above, were immunoblotted with rabbit α-full length ubiquitin at a dilution 1:1000 in 5% non-fat dry milk in TBST for 2 h at RT. Then, incubation with goat anti-rabbit HRP-conjugated antibody at dilution 1:20,000 for 2 h at RT and chemiluminescent detection were performed, as described above.

Immunocytochemistry

Fibroblasts cultured on glass coverslips and iPSC, NES cells and neurons cultured on glass coverslips covered with appropriate cell culture coating were fixed for 20 min with 4% formaldehyde in phosphate-buffered saline (PBS). This was followed by incubation with 50 mM NH₄Cl in PBS for 10 min at RT, permeabilization with ice-cold 0.1% Triton X-100 in PBS for 5 min, and blocking with 5% fetal bovine serum in PBST (PBS with 0.05% Tween-20) for 30 min. Incubations with primary antibodies were performed for 1 h at RT. Fibroblasts were incubated with antibodies against γH2AX, PS1-FL, and BRCA1(Ser1524), iPSC with antibodies against Oct3/4, Nanog and Tra1-60, NES cells with antibodies against Nestin, PAX6, PS1-FL, BRCA1(Ser1524) and amyloid-β, and neurons with antibodies against Tuj3, VGLUT1, VChAT, MAPT, NFL, GFAP, PS1-FL, BRCA1(Ser1524), and amyloid-β (Supplementary Table 5). After 5 washings for 5 min each with PBST, the coverslips were incubated with secondary antibodies for 1 h at RT. Alexa Fluor^®-488-conjugated goat anti-mouse, Alexa Fluor^®-555-conjugated goat anti-rabbit antibody and Alexa Fluor^®-647-conjugated chicken anti-goat antibody (Thermo Fisher Scientific, Invitrogen) at 1:1000 dilution were used. For DNA staining DAPI was used at 1 μg/ml (Sigma-Aldrich) for 10 min at RT. Visualization was performed with Zeiss 780 microscope at the Laboratory of Advanced Microscopy Techniques, Mossakowski Medical Research Centre, or Zeiss Axio Fluorescence Imager at the Department of Neuroscience, Karolinska Institutet. Colocalization was measured with Coloc2 module in the Fiji J software.

Statistical analysis

Bioinformatic statistics was performed in R-CRAN, as described above. The sample size for RNA-seq studies was estimated using ‘ssizeRNA’ R-CRAN package [27]. According to predictions for sequencing depth of 20 million of paired-end reads the minimal sample size was n = 14 provided statistical power at 0.84, reaching recommended 0.8. The sample size estimation was performed at FDR≤0.05, power≥0.8, average read count for each gene in control group = 10; dispersion parameter for each gene = 0.1; fold change for each gene≥2 and proportion of non-DE genes (pi0) = 0.8. All presented graphs were performed and statistics were calculated in Origin Pro 8.0 (OriginLab, USA) and using Microsoft Office Excel. Bar graphs or data points of western blot, immunoprecipitation and ubiquitination assay results are presented as means±SEM of n observations (stated in the figure’s legend). Statistical significance was determined at the 95% confidence level either by one-way ANOVA or repeated measures one-way ANOVA followed by post hoc Tukey test as stated at figure’s legend, defining differences as statistically significant (*p < 0.05; **p < 0.01).

RESULTS

Transcriptome of fEOAD-PSEN1 patients’ fibroblasts reveals dysregulation in CCC and DDR

We performed whole transcriptome profiling of primary fibroblasts derived from fEOAD patients with mutations in PSEN1 compared with neuropsychologically healthy, age- and sex-matched control donors. We detected differentially expressed genes (DEGs) using DESeq2 tool in eight fEOAD-PSEN1 patients carrying mutations A360T, R307S, P267L, L153V, L424R (2 brothers) and I211M (mother and son) versus six controls. The analysis revealed 1228 DEGs, as displayed in the heatmap (Fig. 1A). Using principal component analysis (Fig. 1B) and sample-distance matrix (Fig. 1C), we confirmed that fEOAD samples segregated separately versus controls. This analysis also demonstrated that the I211M family had slightly different transcriptomic profile than the other fEOAD-PSEN1 patients. The volcano plots showed a predominance of upregulated over downregulated genes (Fig. 1D). The dispersion plots showed a general trend of dispersion-mean dependence over the average expression strength (Fig. 1E), and the density analyses revealed that average mean count was equally distributed among the samples (Fig. 1F). Overall, the obtained datasets of DEGs consisted of protein coding transcripts, antisense transcripts and long noncoding RNAs (Supplementary Tables 6–8). In addition, a larger group of sixteen controls compared to the group of listed above eight fEOAD-PSEN1 patients (Supplementary Material Text 1, Supplementary Figure 1) revealed a transcriptional profile similar to that described above. Overall, the most significant DEGs identified by the above analysis were linked with cell cycle checkpoints and DNA damage response in all fEOAD-PSEN1 patients.

Fig.1

Differential gene expression (DGE) analysis of fEOAD versus the highest euclidean distance controls. Whole transcriptome profiling was performed for primary fibroblast cell lines derived from eight fEOAD patients with mutations in PSEN1 (i.e., A360T, R307S, P267L, L153V, L424R_1, L424R_2, I211M_1, I211M_2) and six healthy, age- and sex-matched donors revealed by whole dataset approach shown in Supplementary Figure 1. The DESeq2 comparative analysis of DGE in fEOAD versus control group revealed 1228 differentially expressed genes at fold change >2, FDR <5%, p-value < 0.05, as presented in the heatmap from regularized log transformation counts (A). Principal component analysis showed separate clustering of fEOAD and controls, as well as a distinct expression pattern of the I211M samples (B). The sample-distance matrix showed 6 controls at the highest Euclidean distance from fEOAD, as well as separated clustering of I211M samples (C). The volcano plot showed prevalence of upregulated over downregulated genes (D). The dispersion plots showed a general trend of dispersion-mean dependence over the average expression strength (E), and the density analyses revealed that average mean count was equally distributed among the samples (F). DGE were subjected to the enrichment analyses with IPA tool, revealing a significant enrichment in the cell cycle checkpoints and DDR with a central role of the BRCA1 (G).

In silico functional analysis shows the enrichment in CCC and DDR in fEOAD-PSEN1 patients

By subjecting the obtained list of DEGs to functional bioinformatic analyses, we found a significant enrichment in the signaling pathways and GO terms involved in cell cycle checkpoints, DNA damage response, and proapoptotic signal (Fig. 1G, Supplementary Table 9). All enriched and activated signaling pathways have formed a BRCA1-associated network including “BRCA1 in DNA damage response” or “ATM pathway” (Supplementary Figure 2). Furthermore, selected RNA-seq hits were confirmed by RT-qPCR (Supplementary Figure 3) and by functional studies described below.

DDR induction in fEOAD-PSEN1 patients results in dysregulation of cell cycle and ATM/ATR signaling

Next we have functionally tested DDR in eight fEOAD and six control fibroblast cell lines (same as used for transcriptomic analyses) treated with 2 μM doxorubicin for 0, 6, 16, 24, or 30 h. For the examination of the content of individual proteins, the representative patients’ cell lines (as defined in the legend of the figures, where appropriate) were used. At first, we found that the morphology of fEOAD fibroblasts turned into a senescent phenotype, while majority of control cells remained unchanged (Fig. 2A). Using propidium iodide staining, we observed an increased number of apoptotic cells, reduced number of cells in the G1 phase and increased number of cells in the G2/M phase in fEOAD patients compared to controls (Fig. 2B, C). Moreover, we found that doxorubicin treatment caused an increase in the number of cells in the G1 phase. This increase was significantly higher in fEOAD cells (G1nt-G1doxo = 7.94% cells, n = 6, p = 0.03) than in control ones (G1nt-G1doxo = 4.65% cells, n = 6, p = 0.03). In addition, the number of cells in the G2/M phase increased upon doxorubicin treatment in fEOAD cells, comparing both to non-treated fEOAD cells and to controls (Fig. 2B, C). Furthermore, based on elevated levels of cleaved poly ADP ribose polymerase (cPARP) and of histone H2AX phosphorylated on Ser139 (γH2AX), we confirmed DDR and proapoptotic actions of doxorubicin (Fig. 2D-G). Additionally, following doxorubicin treatment the content of cytosolic yH2AX was higher (Fig. 2H, J), while its nuclear content was lower in fEOAD cells compared to control ones (Fig. 2I, K). Moreover, by immunofluorescence we found that following DDR induction the signal for γH2AX was stronger in the cytosolic compartment in fEOAD cells than in controls, while its nuclear staining pattern was similar in both groups (Fig. 2L). The described above disturbances in cell cycle and DDR in fEOAD fibroblasts were accompanied by abnormalities in the ATM and ATR signaling pathways. This was manifested by an increased content of the ATM kinase phosphorylated on Ser1981 in fEOAD cells compared to controls, together with elevated levels of its major downstream effector, the Chk2 kinase phosphorylated on Thr68 (Fig. 3A). On the contrary, the content of the ATR kinase phosphorylated on Ser428 was lower in fEOAD cells than in control ones under DDR induction, but the content of its downstream effector, the Chk1 kinase phosphorylated on Ser345 was significantly higher in fEOAD cells, both at rest and under DDR induction (Fig. 3B).

Fig.2

Establishing of DDR model of fEOAD and control fibroblasts treated with doxorubicin. Eight primary fibroblasts from fEOAD patients and six controls (same as used in transcriptomic analyses) were treated with 2 μM doxorubicin for 0, 6, 16, 24, or 30 h. For protein content analysis, representative patients cell lines (stated in figure legend) were taken for analysis. Morphology of fEOAD cells turned completely into senescent phenotype, while in controls part of the cells remained intact, as observed with integrated modulation contrast microscopic imaging (Leica Microsystems) (A). Flow cytometry analyses of the apoptosis rate and percentage of cells at G1, S, and G2/M cell cycle phases in eight fEOAD and six controls non treated and treated with doxorubicin for 30 h were conducted with iodide propidium according to Nicolletti’s protocol (B). Flow cytometry data were collected and calculated using CELLQuest Becton Dickinson software. Data represent mean values±SEM. Unpaired-sample Student’s t-test was used for comparison of fEOAD versus control group; p < 0.05, n(fEOAD) = 8 and n(CTRL) = 6 (C). For protein content analysis, representative patients cell lines were taken for analysis. RIPA-total cellular extracts from three fEOAD (L424R_1, R307S, L424R_2) and three control fibroblasts treated for 0, 6, 16, 24, or 30 h with 2 μM doxorubicin (DOXO) were subjected to immunoblotting to verify protein level of cPARP (D), and for γH2AX all fEOAD (R307S, L424R_1, L424R_2, L153V, A360T, I211M_1, I211M_2, P267L, L153V) and all six controls were tested (E). Signal was quantified densitometrically and standardized to GAPDH level (F, G). Cytosolic (H) and nuclear fractions (I) content of γH2AX(Ser139) was estimated in three fEOAD patients (L424R_1, R307S, L424R_2) and three controls and calculated statistically similarly to total protein content (J and K, respectively for each fraction). Data represent mean values±SEM. The selected time point of 30 h as the maximum point of difference between fEOAD versus control cells was based on repeated measures one-way ANOVA with post hoc Tukey test at alpha = 0.05 with significance at *p < 0.05, **p < 0.01. Number of experiments for the content of each protein was at least 3. Each image is a representative of at least three independent experiments. Cellular distribution of γH2AX (Alexa Fluor^®555-red) and DNA damage nuclear foci (DAPI) at rest and upon doxorubicin treatment for 30 h was determined immunocytochemically in all fEOAD and all control cells (L). Microscopic analysis of γH2AX-DNA lesion foci was performed with FijiJ software by 3D object counter (n > 3 images per cell line).

Fig.3

ATM/ATR pathway activation status in fEOAD and control fibroblasts. RIPA-total cellular extracts from fEOAD (R307S, P267L, L424R, L153V, I211M) and corresponding controls treated for 0, 6, 16, 24, or 30 h with 2 μM doxorubicin were subjected to immunoblotting to verify protein level of ATM(Ser1981) and Chk2(Thr68) kinases (A), and ATR(Ser428) and Chk1(Ser345) kinases (B). Signal was quantified densitometrically and standardized to GAPDH level. Data represent mean values±SEM. The difference at selected time points of doxorubicin treatment between fEOAD versus control cells were assessed using repeated measures one-way ANOVA with post hoc Tukey test at alpha = 0.05 with significance at *p < 0.05, **p < 0.01. Number of experiments for the content of each protein was at least 3 (B). Each image is a representative of at least three independent experiments.

DDR machinery is reorganized in fEOAD-PSEN1 patients-derived fibroblasts

The above was accompanied by abnormal expression of several proteins of the ATM/ATR and DDR signaling in fEOAD cells. We found that DDR in fEOAD fibroblast was manifested by increased levels of cell division cycle phosphatase 25C (Cdc25C) phosphorylated on Ser216 (involved in cell cycle re-entry), claspin (DNA binding protein contributing to checkpoint cell cycle arrest), and microcephalin-1 (involved in condensation of chromosomes) (Supplementary Figure 4A-D). Moreover, fEOAD cells showed a decrease in the content of polo-like kinase 1 (Plk1) phosphorylated on Thr210, what might trigger the G2/M transition (Supplementary Figure 4A, E). Additionally, although the level of ATRIP (ATR-interacting protein) remained intact in the cytosol in fEOAD cells following DDR (Supplementary Figure 4A, F), its nuclear content was significantly increased in these cells compared to controls (Supplementary Figure 4A, G). The RPA32 (Replication protein A 32 kDa subunit) protein level remained unchanged (Supplementary Figure 4A, H-I). Finally, protein level of p53 phosphorylated on Ser15, the major proapoptotic effector of the ATM/ATR pathway, was significantly increased in fEOAD cells compared to controls (Supplementary Figure 4A, J-K).

BRCA1 is cytosolic re-localized and hyperactive in fEOAD-PSEN1 patients-derived fibroblasts

Our most striking observation was strong upregulation of the BRCA1 protein phosphorylated on Ser1524 in fEOAD cells in the cytosol fraction, both at rest and following DDR induction (Fig. 4A, C). We have also found that the levels of the BRCA1(Ser1524) in nuclear fractions was lower in fEOAD cells compared to controls (Fig. 4B, E). In addition, the nuclear fraction of the BRCA1(Ser1524) accounted for ca. 4% of the cytosolic fraction in fEOAD cells, while in controls this content reached nearly 27%, what suggested cytosolic retention of the BRCA1(Ser1524) in fEOAD. Besides, we observed additional immunoblotting pattern corresponding to different types of BRCA1, including isoform missing exon 11 (Δex11) of ca. 3.4 kb size encoding ca. 120 kDa protein, as reported by others [28, 29], and an immunoblotting band of ca. 95 kDa likely corresponding to the portion of the protein dimerizing with the BRCA1-associated RING domain protein 1 (BARD1), as reported before [30, 31]. The content of the BRCA1-Δex11 and BRCA1/BARD1 was significantly higher in fEOAD cells than in controls, especially in the cytosol (Fig. 4A, D) and to a less extend in the nucleus (Fig. 4B, F). Finally, we observed the BRCA1-positive immunostaining pattern above the 220 kDa molecular weight, expected to be polyubiquitinated BRCA1 protein, and its content was higher in fEOAD cells than in controls, especially in cytosolic fractions (Fig. 4A, B). Noteworthy, the described above forms of BRCA1 are essential for its E3 ubiquitin ligase activity. The purity of cytosolic and nuclear fractions was verified by immunoblotting with lamin A and GAPDH, respectively, and no signal from tested protein markers was detected. Finally, confocal imaging revealed that the distribution of BRCA1(Ser1524) in fEOAD cells was scattered in whole cell body, in contrast to control cells displaying focused perinuclear staining pattern (Fig. 5A, top).

Fig.4

BRCA1 phosphorylation and subcellular localization. Cytosolic (A) and nuclear (B) fractions from fEOAD (R307S, L424R_1, L424R_2, L153V, I211M_1, I211M_2, P267L) and control fibroblasts from 0, 6, 16, 24, and 30 h of treatment with 2 μM doxorubicin were subjected to immunoblotting to verify protein level of BRCA1(Ser1524). Next to the 220 kDa band of phopsho-BRCA1, the bands of isoform BRCA1 Dex11 and BRCA1/BARD1 complexes were observed at ca. 100–120 kDa and 95 kDa, respectively. Signal was quantified densitometrically and standardized to GAPDH and lamin A levels, respectively for cytosolic (C, D) and nuclear fractions (E, F). Ratio of nuclear to cytosolic fraction was calculated and expressed as % of nuclear portion from cytosolic and nuclear fractions together, giving for 0, 6, 16, 24, and 30 h of 2 μM doxorubicin treatment in [% ]: 28.42, 37.28, 26.67, 3.75, and 15.51 for controls and 3.84, 0.88, 2.53, 11.06, and 1.93 for fEOAD.Data represent mean values±SEM. The difference at selected time points of doxorubicin treatment between fEOAD versus control cells were assessed using repeated measures one-way ANOVA with post hoc Tukey test at alpha = 0.05 with significance at *p < 0.05, **p < 0.01. Number of experiments for the content of each protein was at least 3 (B-K). Each image is a representative of at least three independent experiments.

Fig.5

BRCA1 and PS1 colocalization and coimmunoprecipitation. Cellular distribution of BRCA1(Ser1524) (Alexa Fluor^®555-red) and PS1 (Alexa Fluor^®488-green) at rest and upon treatment with 100 nM bortezomib was determined immunocytochemically (A). Total cellular extracts (immunoprecipitation inputs) from fEOAD and control fibroblasts were incubated with protein G agarose beads and with anti-PS1 antibody and subjected to immunoblotting for BRCA1(Ser1524) (B) and Student’s t-test was used for comparison of control cells with fEOAD at *p < 0.05, for n = 3 (C). Total cellular extracts from fEOAD (R307S, L424R_1, L424R_2, L153V) and controls cells at rest and treated with 50 nM or 100 nM for 24 h bortezomib were subjected to immunoblotting to verify protein level of PS1 FL, PS1 NT, nicastrin (NCST), AβPP and BRCA1(Ser1524) (D). Signal was quantified densitometrically and standardized to GAPDH level (E-L). Data represent mean values±SEM. The difference between fEOAD versus control cells treated at the given concentration of bortezomib were assessed using one-way ANOVA with post hoc Tukey test at alpha = 0.05 with significance at *p < 0.05, **p < 0.01. Number of experiments for the content of each protein was at least 3 (B-K). Each image is a representative of at least three independent experiments.

Upregulation of the BRCA1 protein is accompanied by abnormal distribution and ubiquitination of PS1 in fEOAD-PSEN1 patients-derived fibroblasts

Abnormal subcellular distribution of BRCA1 was accompanied by similarly dispersed immunostaining of PS1 (Fig. 5A, top). Thus, we tested possible interplay between BRCA1 and PS1 by a co-immunoprecipitation using PS1-FL as a bait and immunoblotting resultant precipitates against BRCA1(Ser1524). We observed a significantly higher immunolabeling at 220–250 kDa in fEOAD cells than in control ones, which corresponded to a molecular weight of BRCA1 (Fig. 5B, C). Consequently, we tested the influence of BRCA1-proteaosme dependent activity on PS1 degradation and ubiquitination. For this purpose, fEOAD and control cells were incubated for 24 h with 50 or 100 nM bortezomib, recently recognized as an inhibitor specifically targeting BRCA1 proteasomal activity [32]. Confocal imaging revealed that administration of 100 nM bortezomib restored perinuclear localization of BRCA1 and presenilin 1 in fEOAD cells, while in controls there was no change (Fig. 5A, bottom). In addition, the content of PS1-FL, which was initially lower in fEOAD cells than controls, increased upon BRCA1 inhibition (Fig. 5D, E). At the same time, the content of active PS1 N-terminal fragment (PS1-NT) was significantly lower in fEOAD compared to control. Bortezomib treatment only slightly improved and restored active form PS1-NT in fEOAD cells. Conversely, the amount of PS1-NT has substantially increased upon BRCA1 inhibition in control cells (Fig. 5D, F). This suggests that overexpression and presumably overactivity of BRCA1 could interfere with production and normal functions of PS1. This could also interfere with the assembly of the γ-secretase complex. Consistently, the levels of mature nicastrin, its premature unglycosylated forms and degradation products were higher upon BRCA1-proteasomal inhibition in fEOAD than in controls (Fig. 5D, G-H). Moreover, the level of AβPP and its degradation products increased upon bortezomib treatment to a greater extend in fEOAD cells than in controls (Fig. 5D, I-J). Accordingly, ELISA measurements showed that the production of toxic Aβ42 peptides was higher in fEOAD cells compared to controls (Supplementary Figure 5). Finally, we found that the BRCA1 protein itself accumulated as a result of bortezomib treatment and this effect was more pronounced in fEOAD cells (Fig. 5D, K-L). Altogether, the above data suggest that functionality of the γ-secretase complex and production of Aβ₄₂ peptides in fEOAD cells might depend on proteasomal activity of BRCA1.

DDR interferes with Aβ processing in fEOAD-PSEN1 patients-derived fibroblasts

The described above aberrations associated with BRCA1-proteasomal activity were accompanied by DDR induced Aβ pathology. Namely, DDR induction in fEOAD fibroblasts increased the content of PS1-FL and reduced the content of active PS1-NT and nicastrin (Supplementary Figure 6A-D). In addition, the level of PEN-2, another component of the γ-secretase complex, was also elevated under DDR induction in fEOAD cells compared to controls (Supplementary Figure 6A, E). Lastly, the AβPP content increased in fEOAD cells compared to control following DDR induction (Supplementary Figure 6A, F). Overall, this suggests that abnormal activity of BRCA1 accompanied by DDR stress could mediate degradation of the PS1 or disturb protein turnover of components of the γ-secretase complex, what consequently could affect amyloid processing.

DDR induction is accompanied by increased ubiquitination of BRCA1 and PS1 in fEOAD-PSEN1 patients-derived fibroblasts

In a view of the above-described experiments with the use of bortezomib and based on RNA-seq predictions of the activation of the ubiquitination proteasome system (Supplementary Figure 7), we studied the ubiquitination status of BRCA1 and PS1. We found that following DDR, the BRCA1 protein was ubiquitinated to a greater extend in fEOAD fibroblasts than in control ones (Fig. 6A, C). Moreover, the immunoprecipitates of total BRCA1 analyzed by immunoblotting with antibody against BRCA1(Ser1524) confirmed an increased content of BRCA1(Ser1524) in fEOAD fibroblasts, in particular following DDR (Fig. 6B, D). Furthermore, we observed an enhanced ubiquitination of presenilin 1, both endogenously (Fig. 7A, D) and upon DDR induction (Fig. 7B, E). We also found that fEOAD fibroblasts had a significantly higher degree of total ubiquitination (Fig. 7C, F). This suggests that altered ubiquitination could contribute to the overall accumulation or incorrect sequestration of many different proteins in AD.

Fig.6

Ubiquitination assay: BRCA1. Total cellular extracts (immunoprecipitation inputs) from fEOAD (R307S, L424R_1, L424R_2, P267L) and control fibroblasts from 0, 6, 16, 24, or 30 h of the treatment with 2 μM doxorubicin were incubated with protein G agarose beads and with total BRCA1 antibody (R&D Systems) and subjected to immunoblotting for ubiquitin (A) or BRCA1(Ser1524) (B). Signal was quantified densitometrically and standardized to protein input level. Data represent mean values±SEM. The difference at selected time points of doxorubicin treatment between fEOAD versus control cells were assessed using repeated measures one-way ANOVA with post hoc Tukey test at alpha = 0.05 with significance at *p < 0.05, **p < 0.01. Number of experiments for the content of each protein was at least 3 (C, D). Each image is a representative of at least three independent experiments.

Fig.7

Ubiquitination assay: PS1. Total cellular extracts (immunoprecipitation inputs) from fEOAD (A360T, L153V, R307S, L424R_1, L424R_2, P267L, I211M, I211M_s) and control fibroblasts were incubated with protein G agarose beads and with anti-PS1-FL antibody and subjected to immunoblotting for ubiquitin or PS1-FL. Co-immunoprecipitations of PS1-ubiquitin were performed for non-treated cells (A), or treated for 0, 6, 16, 24, and 30 h with 2 μM doxorubicin to induce DDR (B), revealing increase in ubiquitination rate of presenilin 1 in fEOAD cells in comparison to controls. Total cellular extracts from controls and fEOAD cells treated with doxorubicin were immunoblotted for whole pool of ubiquitin (C). Signal was quantified densitometrically and standardized to protein input level (D-F). Data represent mean values±SEM. One-way ANOVA was used for comparison of fEOAD with control cells under non-treated conditions; *p < 0.05, **p < 0.01, n = 3 (A, D). The difference at selected time points of doxorubicin treatment between fEOAD versus control cells were assessed using repeated measures one-way ANOVA with post hoc Tukey test at alpha = 0.05 with significance at *p < 0.05, **p < 0.01. Number of experiments for the content of each protein was at least 3 (E, F). Each image is a representative of at least three independent experiments.

DDR and BRCA1 signaling is altered in AD iPSC-derived neuronal cells

Next we validated the suggested above role of BRCA1 in neurodegeneration in iPSC-derived neurons and neural stem cells. We used NES cells and 14-days-old neurons derived from fEOAD patient with R307S mutation in PSEN1 and C9Orf72 r(GGGGCC)_n. Among all subjects, the patient was characterized by the fastest progressive and the strongest neurodegeneration leading to severe dementia and death at the age of 63.

iPSC-derived cells reveal proper neuronal characteristics

iPSC (Supplementary Figure 8A-C) have been tested for the expression of pluripotency markers OCT4, KLF4, CMYC and NANOG (Supplementary Figure 8D, E) and SEV encoding Sendai virus protein (Supplementary Figure 8E). We performed immunocytochemical stainings of pluripotency markers: Oct4, Nanog, Klf4 and TRA-1-60 (Supplementary Figure 8F-H). iPSC induced into NES cells were characterized by the presence of neural tube-like rosettes, immunopositive for PAX6 and nestin (Fig. 8A). NES cells exhibited potential to differentiate into neurons upon removal of mitogens (EGF, bFGF) and in the presence of NGF for at least 14 days. Differentiated neurons were immunoreactive for Tuj3, VGLUT1, and VChAT (Fig. 8B). NES cells and neurons were tested for mRNA expression of markers of neural precursors, maturing and mature neurons, and glial cells. The obtained neurons represented a mixed population of maturing glutamatergic, cholinergic and GABAergic neurons. The neurons expressed high levels of DCX (immature neurons & growth cone marker), MAPT, NFL, TUBB3, NCAM (early neuronal markers), and moderate expression of glutamatergic, cholinergic and GABAergic markers, i.e., VGLUT1, GLUL, NMDAR1, CHAT, and GABA (Fig. 8C). By immunoblotting we confirmed that the obtained neurons were positive for microtubule associated protein tau (MAPT) and neurofilament L (Fig. 8D). Finally, we found only few glial cells, as shown by GFAP expression at the detection level, compared to no template control (NTC) (Fig. 8C).

Fig.8

Validation of NES cells and neurons. NES cells grown into neural tube-like rosettes formations and were characterized immunocytochemically for the presence of neural stem cells markers nestin (Alexa Fluor^®555-red) and PAX6 (Alexa Fluor^®488-green) (A). NES cells were differentiated into neurons upon removal of mitogens (EGF, bFGF) and in the presence of neurogenic factors (NGF, BDNF) for 14 days. Differentiated neurons were characterized immunocytochemically for the presence of neuronal markers Tuj3 (Alexa Fluor^®488-red), VGLUT1 (Alexa Fluor^®555-red) and VChAT (Alexa Fluor^®647-cyan) (B). Nuclei were stained with DAPI (blue). Neurons were validated versus NES cells for the presence of neuronal markers at mRNA level using RT-qPCR (C) and at protein level using western blot (D). Neuronal differentiation of NES cells was performed in 3 independent experiments.

Study on iPSC-derived AD neuronal cells confirms abnormalities in DDR and BRCA1 signaling

For DDR induction, NES cells were treated with 2 μM doxorubicin for 0, 6, 16, 24, or 30 h and neurons for 0 or 6 h (Fig. 9). Longer incubation of neurons with doxorubicin resulted in significant cell death, thus very little material could be collected for analysis of RNA or protein. AD and control NES cells showed similar sensitivity to doxorubicin, as demonstrated by the percentage of dying cells stained with Trypan blue and by a loss of neural tube-like rosettes (Fig. 9A, B). AD and control neurons showed similar decline in the number of cells upon doxorubicin treatment (Fig. 9C). In contrast, AD neurons differed from control ones by significantly shorter combined length of neurites (Fig. 9D). We then found that the content of the BRCA1(Ser1524) increased after 6 and 16 h of doxorubicin treatment in AD NES cells, while in controls it was below detection level (Fig. 10A, B). In addition, the content of BRCA1(Ser1524) was significantly higher under both basal conditions and post-DDR induction in AD neurons than control ones (Fig. 10A, C). BRCA1 Δex11 and BRCA1/BARD1 showed similar pattern to that of BRCA1(Ser1524) (Fig. 10A). Furthermore, NES cells contained significantly higher levels of γH2AX than neurons, but only in AD neurons it was significantly higher than in control (Fig. 10A, D, E). DDR induction was accompanied by activation of two BRCA1-related kinases, pATM(Ser1981) and pChk2(Thr68), content of which was significantly increased only in AD NES cells compared to control ones (Fig. 10A, F, H). In contrast, there was no differences in the content of ATM(Ser1981) and Chk2(Thr68) between AD and control neurons (Fig. 10A, G, I). Also, the content of Cdc25C(Ser216), one of the major co-players of BRCA1 in cell cycle re-entry, was significantly elevated in AD neurons, both at rest and following DDR, comparing to control neurons (Fig. 10A, J). Additionally, the p53 protein phosphorylated on Ser15 was detected only in NES cells and its content after doxorubicin was significantly higher in AD NES cells than in control ones (Fig. 10A, K). No phospho-p53(Ser15) was detected in neurons (Fig. 10A). Finally, immunocytochemical staining pattern of BRCA1(Ser1524) significantly differed between in AD and control neuronal cells. Namely, there was significantly higher number of AD NES cells and neurons positive for BRCA1(Ser15324), localization of which correlated with immunostaining of PS1 at significantly higher level in AD than in control cells (Fig. 11).

Fig.9

DNA damage response in NES cells and neurons. Alzheimer’s disease (R307S) and control NES cells were treated with 2 μM doxorubicin for 0, 6, 16, 24, and 30 h (A), cell death was measured by staining with Trypan blue and no difference was found between patient’s and control cells (B). Differentiated neurons were incubated with 2 μM doxorubicin for 0 and 6 h (C), and combined neurite length was counted in ca. 600 cells (D). Neuronal differentiation of NES cells was performed in 3 independent experiments.

Fig.10

BRCA1 and DDR signaling in NES cells and neurons upon doxorubicin treatment. Total cellular extracts from Alzheimer’s disease (R307S) and control NES cells and neurons treated with 2 μM doxorubicin as described in Fig. 9 were subjected to immunoblotting for BRCA1(Ser1524), ATM(Ser1981), Chk2(Thr68), H2AX(Ser139), Cdc25C(Ser216), and p53(Ser15) (A). Signal was quantified densitometrically and standardized to GAPDH level. Data represent mean values±SEM. The difference at selected time points of doxorubicin treatment between AD versus control cells were assessed using repeated measures one-way ANOVA with post hoc Tukey test at alpha = 0.05 with significance at *p < 0.05, **p < 0.01, n = 3 (B-K).

Fig.11

Immunocytochemical staining pattern for BRCA1(Ser1524) and presenilin 1 in NES cells and neurons. Alzheimer’s disease (R307S) and control NES cells (A) and neurons were characterized immunocytochemically for subcellular distribution and colocalization of BRCA1(Ser1524) (Alexa Fluor^®555-red) and PS1 (Alexa Fluor^®488-green) (B). Nuclei were stained with DAPI (blue). BRCA1-PS1 colocalization was assessed by Mander’s factor. Data represent mean values±SEM. The difference between AD versus control cells were tested using one-way ANOVA at alpha = 0.05 with significance at *p < 0.05, n = 3.

Protein ubiquitination is affected in iPSC-derived AD neuronal cells

Following the increased content of different types of BRCA1 related to its E3 ubiquitin ligase activity described above, we found that AD-derived NES cells and neurons displayed higher total level of protein ubiquitination than controls, both before and following doxorubicin treatment (Fig. 12A-C). Next, the immunoblots for ubiquitin were probed for BRCA1, which showed that enhanced ubiquitination correlated with elevated levels of the BRCA1/BARD1 complex in AD cells, especially in neurons (Fig. 12A, D, E). This was accompanied by altered processing of PS1 and AβPP. Endogenous full length presenilin 1 (PS1-FL) was present in control NES cells but was undetectable in AD NES cells (Fig. 13A, B). Upon doxorubicin treatment, the level of PS-FL initially increased and then fell below the detection limit in control NES cells, while in AD NES cells it was undetectable (Fig. 13A, B). However, DDR induction generated some products of PS1 degradation (ca. 37 kDa), with a weak signal in control NES cells, but more pronounced in AD NES cells (Fig. 13A, C). In neurons, PS1-FL level was slightly higher in AD neurons than in control ones at rest, but lower upon doxorubicin treatment (Fig. 13A, D). PS1 degradation products were present in control and AD neurons before doxorubicin treatment and their amount increased after DDR induction. Interestingly, the cleavage pattern of PS1 was different in neurons than in NES cells. Before doxorubicin treatment, the 28 kDa PS1-NT was present only in AD neurons, but its amount increased to similar levels under DDR induction both in AD and control neurons. An additional band of ca. 26 kDa appeared in both AD and control neurons following doxorubicin action (Fig. 13A, E). In contrast, AβPP was present at a substantial level in untreated control and was undetectable in AD NES cells. Upon doxorubicin treatment, AβPP reached similar level in control and AD NES cells, albeit with strikingly different kinetics. In non-treated neurons, AβPP was hardly detectable in both control and AD, while DDR induction resulted in a significant increase of AβPP content only in control neurons (Fig. 13A, F-G). These data on PS1 and AβPP levels underscore differences between NES cells and neurons, likely reflecting their different developmental status. Crucially, the absence of intact AβPP in AD neurons upon DDR induction in contrast to its highly increased content in control neurons suggests its enhanced cleavage and production of toxic Aβ peptides in AD neurons. Accordingly, we found an enhanced immunostaining pattern of Aβ accompanied by abnormal activation of BRCA1 (Supplementary Figure 9). In summary, the above findings demonstrate that dysfunction in amyloid processing in AD neurons upon DDR was accompanied by overactivation of BRCA1.

Fig.12

Protein ubiquitination and BRCA1 status. Total cellular extracts from Alzheimer’s disease (R307S) and control NES cells and neurons treated with 2 μM doxorubicin as described in Fig. 11 were subjected to immunoblotting for ubiquitinated proteins followed by immunoblotting for BRCA1(Ser1524) (A). Signal was quantified densitometrically and standardized to GAPDH level. Data represent mean values±SEM. The difference between AD versus control cells were tested at selected time points of doxorubicin treatment using repeated measures one-way ANOVA with post hoc Tukey test at alpha = 0.05 with significance at *p < 0.05, n = 3 (B-E).

Fig.13

Presenilin 1 and AβPP content and processing upon DDR induction in NES cells and neurons. Total cellular extracts from Alzheimer’s disease (R307S) and control NES cells and neurons treated with 2 μM doxorubicin as described in Fig. 11 were subjected to immunoblotting for presenilin 1 full length (PS1-FL) and N’-terminal fragment (PS-NT) and for amyloid-β precursor protein (AβPP) (A). Signal was quantified densitometrically and standardized to GAPDH level. Data represent mean values±SEM. The difference between AD versus control cells were tested at selected time points of doxorubicin treatment using repeated measures one-way ANOVA with post hoc Tukey test at alpha = 0.05 with significance at *p < 0.05, n = 3 (B-G).

DISCUSSION

Recent studies have suggested that neuronal death in AD may be associated with disturbances in cell cycle checkpoints and DNA damage response. In neurons, this can lead to cell cycle re-entry (CCR) and induce apoptosis [33 –35]. Recently, we have demonstrated robust dysregulation of cell cycle in immortalized lymphocytes derived from fEOAD-PSEN1 patients [36, 37]. It has also been found that postmitotic neurons express a significant number of cell cycle proteins involved in maintenance of genomic stability, including cyclins and cyclin-dependent kinases, neuronal functions of which is either unknown or only partially understood [6, 38]. In this light, we provide important data on the abnormal expression and activation of several proteins associated with CCC and DDR. In addition, we point to the key role of BRCA1 in AD, using three different classes of patients-derived cells: 1) proliferating skin fibroblasts, 2) proliferating neural stem cells, and 3) postmitotic neurons. Overall, we postulate that the BRCA1 protein might be one of the major players in AD, where under DDR stress conditions it can affect PS1 and Aβ processing.

Transcriptomic landscape of dysregulation of CCC and DDR underlies the role of BRCA1

Our transcriptomic data showing strong dysregulation of CCC and DDR complement existing knowledge of the transcriptomic profile of AD patients provided by several studies involving the use of peripheral blood lymphocytes and postmortem brains [39 –42]. Conversely, RNA-seq studies with the use of senescing fibroblasts revealed downregulation of genes involved in DNA synthesis and repair and of most of the cell cycle pathways [43]. This suggests that cell death mechanisms differ during pathological aging and AD. Importantly, our data from fEOAD fibroblasts are consistent with the microarray results from sporadic AD fibroblasts displaying oxidative stress phenotype [44]. Hereby, we present a scenario of how disturbed cell cycle signaling and DDR can promote cell death in AD.

Based on our transcriptomic data derived from fEOAD fibroblasts, the abnormalities in cell cycle checkpoints and DDR signaling in fEOAD cells involved destabilization of centrosomes. Importantly, centrosomal organization is known to be primarily controlled by cytosolic-localized BRCA1 [45, 46]. Moreover, an increased cytosolic sequestration of activated BRCA1 was found to induce apoptosis as a result of disturbed cell cycle in the G2/M phase [47]. Furthermore, centrosomal stability, ensured in the G2/M checkpoint, has been found to involve BRCA1-dependent phosphorylation of the Chk1 kinase, what further induced the Cdc25C protein and the Cdc2/cyclin B kinase complex, promoting cell cycle arrest in S and G2/M phase [48, 49]. Accordingly, we found that fEOAD cells with cytosolic re-localized upregulated BRCA1(Ser1524) were arrested in the G2/M phase. Furthermore, we found that the dynamics of recruitment of BRCA1 and Chk1 during doxorubicin treatment overlapped in fEOAD fibroblasts. Thus, we propose that upregulated cytosolic BRCA1(Ser1524) protein may adversely affect genome integrity in AD by mediating the G2/M arrest through the phosphorylation of Chk1 followed by the activation of the Cdc25C protein. Furthermore, centrosomal regulation of the G2/M checkpoint involves the activation of Chk2 kinase, its upstream regulator ATM kinase and p53 protein, which mediates phosphorylation of BRCA1 following an induction of mitotic regulators, such as aurora A and polo-like kinases (Plk1 and Plk3). Endorsing the suggested BRCA1-driven block in the transition of the G2/M phase, we observed an increase in the content of Plk1 upon DDR induction in fEOAD cells. Furthermore, claspin, known to regulate Chk1 activation, formation of Chk1-BRCA1 complex and Chk1-dependent phosphorylation of BRCA1 at Ser1524 [50], was found to be upregulated in fEOAD cells, both at rest and following DDR. All this indicates the involvement of BRCA1 in the G2/M checkpoint dysfunction in AD. Furthermore, DNA damage might also activate the G1/S cell cycle checkpoint and cell cycle arrest. Accordingly, we have observed an increased number of fEOAD cells arrested at the G1/S phase, but in contrast to the G2/M, only following DDR. BRCA1 was shown to facilitate p53 phosphorylation by the ATM kinase during DDR, resulting in a p53-dependent induction of the p21 protein and G1/S phase arrest [51]. Consistent with this, our recent studies on fEOAD-derived lymphocytes showed elevated content of p53 (Ser15), which led to increased cytoplasmic expression of p21 under apoptosis induction with 2-deoxy-D-ribose [37]. Also, with the use of fEOAD-fibroblasts, we have observed an increased content of the p53(Ser15) protein following DDR. This was accompanied by upregulation of phosphorylated forms of the ATM and Chk2 kinase following DDR. The above data suggest the need for signaling at the BRCA1-ATM/Chk2/p53/p21 axis for cell cycle arrest in the G1/S phase. It can be further speculated whether activation of this axis leads to dysregulation of asymmetrical divisions of neural precursors during adult neurogenesis, a disturbance of which has been recently suggested [52]. All described above dependencies between various players of cell cycle checkpoints are illustrated in the scheme (Fig. 14). In summary, BRCA1 has been implicated in the activation of different cell cycle checkpoints in order to allow DNA repair before resuming the cell cycle or guiding the cells to death [53]. Based on all above, we postulate that overactive BRCA1 in AD patients’ cell lines might contribute to both G1 and G2 arrest following DDR stress and further trigger cell death.

Fig.14

DNA damage response and activation of cell cycle checkpoints: a possible link with Alzheimer’s disease. Key players of ATM and ATR signaling pathways regulating DNA repair, apoptosis, aging, cell cycle checkpoints at G1/S phase and G2/M phase are depicted in the scheme. The BRCA1 protein is presented as a central mediator of all of these processes with a major effector being the p53 protein.

Hyperactive BRCA1 implicates cell cycle re-entry in AD-neural cells

The above paragraph describes abnormalities in non-neuronal cells. Importantly, we have observed that BRCA1 and several components of DDR and CCC were also upregulated in neurons and in neural stem cells. In proliferating cells BRCA1 phosphorylation increases in late G1 and early S phase, while upon cell cycle exit it becomes progressively dephosphorylated and its overall level drops [54]. Thus, upregulation of BRCA1 phosphorylated on Ser1524 in AD neurons suggests its role in cell cycle reactivation. This is in agreement with aforementioned concept of cell cycle re-entry in postmitotic neurons. Furthermore, recent neurodevelopmental studies showed that BRCA1-Δex11 knockout mice had reduced brain volume in all brain layers [6]. These studies suggested that BRCA1 contributes to the apoptosis of neural precursors and its activity should be extinguished to enable proper neuronal maturation. Moreover, the BRCA1 protein was suggested to act as a centrosomal factor in establishing the cellular polarity of neural progenitors through the DNA damage sensor kinase ATM [6]. This is consistent with our findings showing DDR-driven upregulation of BRCA1(Ser1524) in AD NES cells was followed by an activation of the ATM/Chk2 kinases. Noteworthy, in AD neurons the content of phospho-ATM and phospho-Chk2 remained unchanged, despite significant upregulation of BRCA1(Ser1524). Thus, we propose that BRCA1 activation may have different implications at different developmental stages of AD, varying between neural stem cells and neurons. We further postulate that BRCA1 drives CCR and incorrect DDR in AD neurons. This is consistent with the upregulation of BRCA1-Δex11 in AD neurons, known to cause genetic instability due to impaired ubiquitination of BRCA1 targets leading to unrepaired DNA lesions [29]. AD neurons were also characterized by higher levels of γH2AX than control neurons, possibly indicating more extensive DNA damage. This suggests that AD neurons underwent genotoxic stress driven by endogenous factors. Finally, we tested one of possible player of CCR, connected with BRCA1, the Cdc25C phosphatase. Cdc25C activates cyclin dependent kinase 5, which regulates neuronal death evoked by DNA damage [55]. Cdc25C is one of the ubiquitination targets of BRCA1 important during the G2/M cell cycle transition [56]. BRCA1 also plays a role in the G2/M checkpoint via enhanced phosphorylation and altered cellular localization of Cdc25C [49]. Interestingly, we observed an increased content of Cdc25C(Ser216) in AD neurons, both at rest and following DDR. In this regard, ours and others’ studies indicate that endogenous activity of Cdc25 could be important for the regulation of cell cycle-mediated neuronal death. Our findings on the Cdc25C support the CCR concept in AD neurons.

Hyperactive BRCA1 influences PS1 turnover and Aβ processing in fEOAD cells

Next we asked whether abnormal expression of BRCA1 in fEOAD cells could be linked with Aβ pathology. To the upregulation of BRCA1(Ser1534)m we found an increased content of the forms of BRCA1 related to its E3 ubiquitin ligase activity, i.e., Δex11 isoform, BARD1-reactive BRCA1 and poly-ubiquitinated BRCA1, in all types of analyzed fEOAD cells. Consistent with this, numerous reports indicate altered homeostasis of the ubiquitination system in neurodegenerative disorders [56 –58]. Moreover, a tight relationship between Aβ pathology and the ubiquitin-proteasome system has been proposed [59]. Consequently, we observed a significant accumulation of ubiquitinated proteins in all tested fEOAD-derived cell types, both at rest and following DDR. The enhanced total ubiquitination observed already under basal conditions was similar to those found in cells treated with proteasome inhibitors [60, 61]. This is in a line with data showing that ubiquitinated protein aggregates are formed by ‘trapping’ of ubiquitin molecules depleting the free ubiquitin pool [56]. The enhanced total cellular ubiquitination was accompanied by abnormalities in Aβ processing machinery in fEOAD cells. Altered ubiquitination of PS1 brings a question on the importance of the E3 ubiquitin ligase activity of BRCA1 protein. Indeed, we found that BRCA1 colocalized and coimmunoprecipitated with PS1 in fEOAD cells. Importantly, BRCA1 functions differ when localized in different subcellular compartments. For instance, BRCA1 localized in the endoplasmic reticulum was found to trigger apoptosis by interfering with the IP3 receptor/Ca^2 + channel and evoking a massive Ca^2 + efflux [62], while in the mitochondria it was found to interplay with the Bcl-2 protein inducing caspase 3-apoptotic cascade [63]. In our study, we found cytosolic and dispersed immunolabeling pattern both for BRCA1 and PS1 in all types of fEOAD cells. Interestingly, nuclear spots-like staining of PS1 was well pronounced in fEOAD fibroblasts. This is in agreement with data showing nuclear shuttling of PS1 followed by its accumulation in ER in pathological aging [64], and during muscle fibers degeneration with well pronounced inclusions of PS1-ubiquitin complexes [65]. Furthermore, restoring of normal localization of PS1 and BRCA1 upon BRCA1-proteasomal inhibition with bortezomib suggested that the excess of BRCA1 in the cytosol in fEOAD cells may be a source of its increased E3 ubiquitin ligase activity. This may contribute to the ‘trapping’ of PS1 by its improper sequestration when overubiquitinated. Accordingly, it is known that BRCA1 E3 ubiquitin ligase activity redirects various proteins to different subcellular compartments, including proteasomal-lysosomal degradation pathway [5]. This is in agreement with data showing the formation of pathological aggregosomes composed of PS1 and ubiquitin in AD [66]. Additionally, the BRCA1-Δex11, found to be upregulated in our fEOAD samples, is an isoform missing NLS (nuclear localization sequence) portion and thus highly expressed in the cytoplasm [67]. Also, fEOAD cells showed upregulation of cytosolic BRCA1/BARD1 immunoreactive band. Importantly, this dimer when localized in the cytosol is linked with promotion of apoptosis by the ubiquitination of γ-tubulin during centrosome duplication [45]. PS1 was also found to be located at centrosomes [68], mainly through interactions with microtubule-binding proteins such as CLIP-170 [69] and with γ-tubulin [70]. Noteworthy, centrosomes are crucial for coordinating neuronal migration and polarization, contributing to the segregation of cell fate factors, efficient nucleokinesis, and directed neurite outgrowth [71]. Accordingly, we found that the total length of neurites was significantly lowered in AD neurons. It can be assumed that the upregulation of BRCA1/BARD1 in AD neurons could adversely affect centrosomal cytoskeleton-organizing center and destabilize neuronal polarization. Furthermore, based on bortezomib- and doxorubicin-treatment studies, we concluded that overactive BRCA1 might influence not only the turnover and degradation pattern of presenilin 1, but also of nicastrin and AβPP, and thus Aβ processing. Importantly, the level of PS1-FL increased in doxorubicin-treated fEOAD cells, while active PS1-NT dropped. This was observed in both fibroblasts and neuronal cells and suggested abnormalities in AβPP processing. Interestingly, it has been proposed that dysregulation of the ubiquitination-proteasome system serves to shunt AβPP into proteolysis, leading to increased generation of Aβ [72]. All the above is in agreement with reports showing that enhanced ubiquitination of PS1 contributes to the production of toxic Aβ₄₂ peptides [73]. We propose that BRCA1 might influence intracellular traffic and/or degradation of the components of the γ-secretase complex, including PS1, and influence Aβ processing in fEOAD cells.

Consequences of increased levels of BRCA1 in AD neurons

Recently, it has been demonstrated that the level of non-phosphorylated BRCA1 in degenerating neurons with dystrophic neurites and granulovacuolar disintegration in late-onset AD postmortem brains was lower than in control cells. Notably, the remaining yet non-degenerating neurons in the AD brains displayed an elevated content of the BRCA1 protein compared to the control neurons [8]. We found a significantly increased content of BRCA1(Ser1524) in AD-derived neurons of fEOAD patient. We postulate that the prodromal phase of the disease could be triggered by overactive BRCA1, resulting in further BRCA1 self-clearance and depletion at the final stage of the disease, as suggested by Suberbielle and coworkers [8].

Summarizing, in this work we present data indicating that overactivation and cytosolic re-localization of the BRCA1 protein can play a significant role in neuronal death in AD. We suggest that this mechanism could involve enhanced total protein ubiquitination, and incorrect degradation and turnover of presenilin 1, resulting in aberrant AβPP processing and toxic amyloid production. Furthermore, the hyperactive BRCA1 could introduce important signaling for cell cycle re-entry of AD neurons, ultimately leading to their apoptosis. Our findings provide novel insight on BRCA1 activity and function in neurons in AD and open up a promising novel research area.

Footnotes

ACKNOWLEDGMENTS

The authors would like to thank all the patients, their families, and control donors involved in this study. We also thank Ms. Małgorzata Kobryś for DNA isolation from blood samples prior mutation detection and Ms. Anna Piotrowska and Prof. Jan Fronk for editorial help.

The study was supported by the National Science Centre (Poland) (2013/09/D/NZ3/01348 to MW; 2011/02/A/NZ2/00014 to KG; 2015/17/D/NZ2/03711 to MS) and the Foundation for Polish Science (TEAM to KG). Microscopic imaging was performed in the Laboratory of Advanced Microscopy Techniques, Mossakowski Medical Research Centre, Polish Academy of Sciences. Some experiments were carried out at the Nencki Institute Neurobiology Center with the use of CePT infrastructure financed by the European Regional Development Fund Operational Programme “Innovative economy” for 2007-2013.

RNA sequencing data are available at the Sequence Read Archive NCBI NIH database as the BioProject PRJNA382346 ().

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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