amyloid-b toxicity modulates tau phosphorylation through

Amyloid-b toxicity modulates tauphosphorylation through the PAX6signalling pathwayYalun Zhang,1,2,3,4,† Yi Zhang,1,† Yahyah Aman,5 Cheung Toa Ng,1,2 Wing-Hin Chau,1

Zhigang Zhang,1 Ming Yue,1 Christopher Bohm,3,4 Yizhen Jia,1 Siwen Li,1

Qiuju Yuan,6 Jennifer Griffin,3,4 Kin Chiu,7 Dana S. M. Wong,1 Binbin Wang,8

Dongyan Jin,1 Ekaterina Rogaeva,3,4 Paul E. Fraser,3,4 Evandro F. Fang,5

Peter St George-Hyslop4,9 and You-Qiang Song1,2,10

†These authors contributed equally to this work.

The molecular link between amyloid-b plaques and neurofibrillary tangles, the two pathological hallmarks ofAlzheimer’s disease, is still unclear. Increasing evidence suggests that amyloid-b peptide activates multiple regula-tors of cell cycle pathways, including transcription factors CDKs and E2F1, leading to hyperphosphorylation of tauprotein. However, the exact pathways downstream of amyloid-b-induced cell cycle imbalance are unknown.Here, we show that PAX6, a transcription factor essential for eye and brain development which is quiescent inadults, is increased in the brains of patients with Alzheimer’s disease and in APP transgenic mice, and plays a keyrole between amyloid-b and tau hyperphosphorylation.Downregulation of PAX6 protects against amyloid-b peptide-induced neuronal death, suggesting that PAX6 is akey executor of the amyloid-b toxicity pathway. Mechanistically, amyloid-b upregulates E2F1, followed by the in-duction of PAX6 and c-Myb, while Pax6 is a direct target for both E2F1 and its downstream target c-Myb.Furthermore, PAX6 directly regulates transcription of GSK-3b, a kinase involved in tau hyperphosphorylation andneurofibrillary tangles formation, and its phosphorylation of tau at Ser356, Ser396 and Ser404.In conclusion, we show that signalling pathways that include CDK/pRB/E2F1 modulate neuronal death signals byactivating downstream transcription factors c-Myb and PAX6, leading to GSK-3b activation and tau pathology, pro-viding novel potential targets for pharmaceutical intervention.

1 School of Biomedical Sciences, University of Hong Kong, Hong Kong, China2 HKU-Shenzhen Institute of Research and Innovation, University of Hong Kong, Hong Kong, China3 Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON, M5T 0S8, Canada4 Department of Medical Biophysics, and Medicine (Neurology), University of Toronto, Krembil Discovery Tower,

Toronto, ON, M5T 2S8, Canada5 Department of Clinical Molecular Biology, University of Oslo and the Akershus University Hospital, 1478 Lørenskog,

Norway6 School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China7 Department of Ophthalmology, University of Hong Kong, Hong Kong, China8 Department of Genetics, National Research Institute for Family Planning, Beijing, China9 Cambridge Institute for Medical Research, Department of Clinical Neurosciences, University of Cambridge,

Cambridge CB2 0XY, UK10 The State Key Laboratory of Brain and Cognitive Sciences, University of Hong Kong, Hong Kong, China

Received September 21, 2020. Revised March 08, 2021. Accepted March 14, 2021VC The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.For permissions, please email: [email protected]

doi:10.1093/brain/awab134 BRAIN 2021: of 12 | 1

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https://orcid.org/0000-0002-4052-818X

https://orcid.org/0000-0001-9407-2256

Correspondence to: Youqiang SongSchool of Biomedical SciencesUniversity of Hong Kong, Hong Kong, ChinaE-mail: [email protected]

Keywords: Alzheimer’s disease; amyloid-b plaques; neurofibrillary tangles; tau phosphorylation

Abbreviation: ChIP = chromatin immunoprecipitation

IntroductionGenetic and cell biology studies have shown an important role foramyloid-b peptide and microtubule-associated protein tau in thepathogenesis of Alzheimer’s disease.1,2 The molecular pathwayslinking amyloid-b, tau and cell death are controversial.3–5 Some evi-dence from experiments, including in vitro studies of neuronal celldeath, in vivo investigation of animal models, and human post-mor-tem studies, support a hypothesis that implicates deregulation ofcell cycle proteins as key mediators of neuronal dysfunction andloss in Alzheimer’s disease brains.6,7 For example, cell division cycle25 (CDC25) phosphatases have increased expression and activity inAlzheimer’s disease brains.8,9 Multiple cyclin-dependent kinases(CDKs: CDK1, CDK4 and CDK6), retinoblastoma protein (pRB), cyclins(A, B, C, D and E) and CDK inhibitors are overexpressed in cellularand animal models and post-mortem brains of patients withAlzheimer’s disease.10–19 Central to the hypothesis of the involve-ment of cell cycle deregulation in neuronal death in Alzheimer’sdisease is the CDK/pRB/E2F1 pathway, wherein amyloid-b activatesCDK4/6, leading to pRB phosphorylation and activation of E2F1 tran-scription factor. However, the precise detail of the mechanism bywhich amyloid-b accumulation is linked to neurofibrillary tanglespathology in Alzheimer’s disease remains elusive.

Materials and methodsHuman brain specimens and animals

Post-mortem human brain tissues from the frontal cortex ofpatients with Alzheimer’s disease and non-demented control sub-jects were obtained from the Canadian Brain Tissue Bank and NewYork Brain Bank of Columbia University. The study of human braintissue was approved by the Ethics Committee of the Faculty ofMedicine of the University of Toronto (N0: 00026798) and theUniversity of Hong Kong (N0: UW 13–177). All mice were on aC57BL/6 genetic background. TgCRND8 mice express human amyl-oid precursor protein 695 (APP695) with the Swedish mutation(KM670/671NL) and Indiana mutation (V717F) under the control ofthe Prpn (PrP) gene promoter.20 All experiments using animalswere approved by the Committee for the Use of Live Animals inTeaching and Research at the University of Hong Kong (N0:CULATR2792-12, CULATR 3732-15, CULATR 5212-19).

Antibodies and reagents

For western blot, anti-Pax6 (Thermo Fisher Scientific, 42-6600,1:1000), anti-Pax6 (Sigma, SAB5300039, 1:1000), anti-c-Myb (CellSignaling, 12319, 1:1000), anti-c-Myb (Abcam, ab117635, 1:3000), anti-E2F1 (Santa Cruz, sc-251, 1:1000), anti-GSK-3b (Cell Signaling, 9315,1:3000), anti-phospho-Tau (pSer356) (Sigma, SAB4504556, 1:1000),anti-phospho-Tau (pSer396) (Sigma, T7319, 1:3000), anti-phospho-Tau (pSer404) (Sigma, T7444, 1:3000), anti-b-actin (Cell Signaling,4970, 1:3000), and anti-GAPDH (Cell Signaling, 2118, 1:3000) were

used. For chromatin immunoprecipitation, anti-Pax6 (Santa Cruz,sc-32766, 1:50), anti-c-Myb (Santa Cruz, sc-74512, 1:50), anti-E2F1(Santa Cruz, sc-251, 1:50), and anti-Flag (Sigma, F1804, 1:500) wereused. For immunostaining, anti-Pax6 (Covance, PRB-278P, 1:2000),anti-NeuN (Chemicon, MAB377, 1:1000), secondary antibodies goatanti-rabbit Alexa FluorVR 568 (Thermo Fisher Scientific, A-11011,1:4000) and goat anti-mouse Alexa FluorVR 488 (Thermo FisherScientific, A-11008, 1:4000) were used. For primary neuron treat-ment, amyloid-b1-42 peptide was purchased from Bachem (H1368),and flavopiridol was purchased from Sigma (F3055).

Plasmid constructs

PAX6 P1 promoter luciferase reporter construct pGL3b-Pax6 (1–346)was a gift from Dr Andrew Chantry; PAX6 luciferase reporter con-struct P6CON-luc, PQ107 was a gift from Dr Ales Cvekl; c-Myb shRNAconstruct pSiren-retroQ-Myb and control construct pSiren-retroQ-Luc were gifts from Dr Robert K. Slany; c-Myb responsive reporterconstruct p5xMRE-A-luc (5xMRE) and wild-type c-Myb constructpcDNA3.1-FL-c-Myb were gifts from Dr Juraj Bies; E2F1 shRNA con-struct BS/U6 E2F1 RNAi and control construct BS/U6 RNAi vectorwere gifts from Dr W. Douglas Cress; and wild-type E2F1 constructpcDNA3.1-HA-E2F1 was a gift from Dr Joseph R. Nevins.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) experiments were con-ducted as previously described.21 Mouse cortical neurons andHEK293 cells were harvested and cell lysates were sonicated andcentrifuged, and the supernatants collected for immunoprecipita-tion. Brain samples from 10 Alzheimer’s disease cases and 10 con-trols were obtained from the University of Columbia. Equalamounts (30–40 mg) of frozen tissue from each sample wereweighed and thawed on ice. Tissues were chopped with a scalpeland transferred to conical tubes with ice-cold PBS containing pro-tease inhibitor cocktail (Sigma). Tissues were cross-linked with 1%formaldehyde in PBS for 10 min at room temperature and 125 mMglycine was added to quench the reaction. Tissues were centri-fuged at low speed (1300 rpm) at 4�C for 5 min and the supernatantwas removed. The pellets were washed with ice-cold PBS twice,centrifuged and resuspended in lysis buffer (1% SDS, 10 mM EDTA,50 mM Tris/HCl, pH 8.1) containing protease inhibitor cocktail(Sigma). Next, pellets were homogenized with a Dounce tissuegrinder pestle in 20 strokes, and lysates were sheared by sonic-ation for 50–100 s in 10 s bursts. Samples were centrifuged at 4�Cfor 10 min at 15 000 rpm to remove debris, and supernatants werediluted 1:10 in a lysis buffer (0.01% SDS, 1% TritonTM X-100, 2 mMEDTA, 20 mM Tris/HCl, pH 8.1, 150 mM NaCl). An aliquot of 20 mlfrom each undiluted sample was stored for the input chromatin.Immunoprecipitations were performed with specific antibodies at4�C overnight with rotation, a non-specific anti-Flag antibody wasused as a negative control. ChIP-Grade Protein G Magnetic Beadsslurry (30 ll) (#9006, Cell Signaling) was added to each sample and

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then incubated with rotation for 2 h. Beads were pelleted using amagnetic separation rack and washed four times with washingbuffer. Beads were eluted twice with elution buffer (1% SDS, 0.1 MNaHCO3) by rotation at 30�C for 15 min. Formaldehyde cross-link-ing was reversed by overnight heating at 65�C, as well as input,incubating for 1 h with RNase A at 37�C (20 mg/ml) and then for 1 hwith proteinase K at 42�C (20mg/ml). DNA was purified using a PCRpurification kit (Qiagen), and samples were analysed by semi-quantitative PCR.

Mining human brain microarray datasets

The workflow of data processing is shown in Supplementary Fig.1A. Four steps were performed to screen and rank genes regulatedby E2F1. First, gene expression profile data for GSE1522222 wereobtained from the Gene Expression Omnibus (GEO) database (plat-form GPL2700). Brain samples with a confirmed pathological diag-nosis of late-onset Alzheimer’s disease (n = 176) and control brains(n = 187) were extracted. Gene differential expression was assessedby R package Limma (version 3.40.6),23 and genes with fold change41.5 and adjusted P-value 50.05 were categorized as differentiallyexpressed genes. ChIP-sequencing datasets for human transcrip-tion factor binding sites were downloaded for annotation andscreening of the downstream targets of E2F1.24 Potential E2F1downstream regulatory genes were derived by overlapping E2F1transcription factor binding sites and the promoter regions (2 kbupstream to 1 kb downstream of the transcription start site foreach gene) of the differentially expressed genes. Gene markers inthe human brain were extracted from the Cell Marker database25

for further screening. Finally, Gene Ontology (GO) semantic simi-larity scores of the selected genes were assessed by R packageGOSemSim (version 2.10.0).26 The overall relevance between E2F1and the selected genes was calculated based on the arithmeticaverage value of the three GO semantic similarity scores (biologicalprocess, molecular function and cellular component).

RNA-sequencing

Total mRNA was extracted from cultured mouse primary neuronstransfected with a control siRNA and Pax6 siRNA. For each controlsiRNA or Pax6 siRNA treatment, one biological replicate was pre-pared. All samples were sequenced by Axeq using Illumina HiSeq2x100 bp paired-end sequencing. Eight raw sequence files weregenerated in FASTQ format. The quality of RNA-sequencing rawreads was evaluated using NGS QC Toolkit (version 2.2.3). The totalnumber of reads per sequence file was around 32 million and theaverage read length was 101 bp. The overall PHRED quality scoresfor all sequence files was higher than 20 and the peak value of GCcontent distribution for each sequence file was 45–55%. No furthertrimming or filtering was applied to the raw data.

Mapping RNA-sequencing reads to a mousereference genome using TopHat

Millions of short reads from RNA-sequencing were aligned to themouse reference genome using TopHat (version 2.0.3). TopHat firstapplies an unspliced aligner, Bowtie, to align exon reads to a refer-ence genome without considering any large gaps and then dealswith the small portion of unmapped reads at splicing junctions.27

The mouse reference genome sequence was downloaded from theTopHat website (Mus musculus iGenome Ensembl NCBIM37).TopHat accepts raw reads files in FASTQ format as input and out-puts the alignment results in BAM format. Default parameterswere used to run TopHat. Over 80% of the reads could be mappedto the reference genome.

Aligned reads summarization and transcriptomereconstruction using Cufflinks

Aligned RNA read files in BAM format were inputted into Cufflinks(version 2.0.0) to assemble the aligned reads into transcripts.Cufflinks uses a reference genome-guided method to assembleexons, identify novel transcripts and report a minimal set of iso-forms to best describe the reads in the dataset.28 Cufflinks normal-izes the raw fragment counts with a maximum likelihoodestimation method and quantifies the expression of transcripts asa fragments per kilobase of exon model per million mapped frag-ments (FPKM) value. Cuffmerge was used to merge all transcriptassemblies in GTF format to construct a more complete and accur-ate transcriptome. This transcriptome was then compared withthe mouse reference annotation file downloaded from Ensembl(NCBIM37) to identify novel genes and transcripts byCuffcompare.29

Differential expression test using Cuffdiff andcandidate gene selection

Cuffdiff, which is included in the Cufflinks package, was used todetect differentially expressed genes or transcripts between thenormal control and the Pax6 knockdown groups in the mouseRNA-sequencing experiment. Differential gene expression afterPax6 knockdown was calculated by the ratio of FPKM value at P0versus C0. In total, 8584 genes (4520 downregulated and 4064 upre-gulated) were identified as differentially expressed with false dis-covery rate-adjusted P-values 5 0.05. Pathway enrichmentanalysis based on these differentially expressed genes identified60 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG)pathways with P-values 5 10–4. Alzheimer’s disease-related KEGGpathways were defined as pathways that share at least one genewith the Alzheimer’s disease pathway hsa05010; 77 Alzheimer’sdisease-related KEGG pathways were identified. As a result, 34Alzheimer’s disease-related enriched KEGG pathways were chosenand genes involved in these pathways were downloaded from theKEGG database.30 We searched the promoter regions of all differ-entially expressed genes for PAX6 binding sites using a PAX6 bind-ing matrix from JASPAR. Finally, 33 genes were selected that metall of the following criteria: 41.2 expression fold change, predictedto have PAX6 binding sites and involved in at least threeAlzheimer’s disease-related enriched KEGG pathways(Supplementary Table 1).

Primary neuronal cultures and RNA interference

Primary cortical neurons were derived from embryonic Day 14.5(E14.5) C57BL/6 mice.31 The brains were dissociated from skulls,meninges removed and cortices isolated. Individual cells were dis-sociated by incubation with trypsin and DNase (Sigma). Cells wereplated on poly-D-lysine (Sigma) coated 24-well plates at a densityof 1.5 � 106 cells/ml in NeurobasalTM medium (Thermo FisherScientific) supplemented with B-27 (Thermo Fisher Scientific), N-2(Thermo Fisher Scientific), and glutamine (0.5 mM, Thermo FisherScientific). Three days after seeding, cultured cells were trans-fected with siRNAs (60 pmol/24-well) using LipofectamineVR 2000transfection reagent (Thermo Fisher Scientific). Twenty-four hoursafter transfection, cells were exposed to amyloid-b1-42 (5mM) forthe indicated time periods and then processed for subsequentassays. SiRNAs from Santa Cruz are provided as pools consistingof three to five target-specific 19–25 nucleotides siRNAs designedto specifically knockdown the expression of mouse PAX6, c-Myb orE2F1. SilencerVR Select pre-designed siRNAs were from Ambion,non-targeting siRNA was used as a negative control. Knockdownefficiency per target was tested by RT-PCR or western blot.

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Luciferase reporter assay

Cortical neurons were transfected with luciferase reporterconstructs three days after plating. After amyloid-b treatment,luciferase activity of a target gene was determined using the dual-luciferase reporter assay system (Promega) in a microplate lumin-ometer. Luciferase activity was normalized against Renilla activityfor the transfection efficiency using a pRL Renilla luciferase controlreporter construct.

Western blot analysis

Human brain tissues or mouse cortical neurons were dissolved inice-cold 1� RIPA buffer (Cell Signaling) with proteinase inhibitorcocktail (Sigma). Proteins were separated on NuPAGETM Novex4–12% Bis-Tris gels (Thermo Fisher Scientific) and transferred tonitrocellulose membranes. Membranes were probed with indi-cated primary antibodies. The blots were analysed and quantifiedby densitometry using ImageJ software (Version 1.52t, National

Institutes of Health).

Immunostaining

TgCRND8 mice and non-transgenic littermate pairs at 2, 4, 6, 8, 13,26 weeks of age were sacrificed by transcardial perfusion with 1�PBS, brains were fixed in 4% paraformaldehyde and cryoprotected.Horizontal sections were cryostat-cut at 10mm thickness from OCTembedded frozen blocks and mounted onto gelatin-coated slides.The sections were blocked with 10% goat serum (Sigma) in antibodydiluent (Dako) for 1 h at room temperature. The sections were incu-bated overnight at 4�C with: Rabbit anti-Pax6 (Covance, 1:2000) andmouse anti-NeuN (Chemicon, 1:1000) antibodies [prepared in anti-body diluent (Dako)], followed by incubation with second antibodiesgoat anti-mouse antibodies (Alexa FluorVR 488, Thermo FisherScientific, 1:4000) or goat anti-rabbit antibodies (Alexa FluorVR 568,Thermo Fisher Scientific, 1:4000) for 1 h. The slides were mountedin a DAPI/Antifade mounting medium (Vectashield). Fluorescentimages were captured using a confocal laser-scanning microscope(LSM 710, Carl Zeiss).

Amyloid-b preparation

Oligomeric amyloid-b1-42 peptide (Bachem) was prepared asdescribed previously.32 Briefly, lyophilized, HPLC-purified amyloid-b1-42 was equilibrated and reconstituted in 100% 1,1,1,3,3,3 hexa-fluoro-2-propanol (HFIP; Sigma) to 1 mM. HFIP was evaporated andthe crystallized peptide was air-dried and was reconstituted to5 mM in dimethyl sulphoxide (DMSO) followed by sonication. The5 mM amyloid-b1-42 stock solution was then diluted with phos-phate-buffered saline (PBS) to 400 mM and was incubated for 18–24 h at 37�C. The solution was diluted again for a working concen-tration of 100 lM. The resulting solution was further incubated at37�C for 18–24 h and the amyloid-b peptide was ready for use.

Semi-quantitative RT-PCR

Total mRNA was prepared from cell cultures utilizing TRIzolVR re-agent (Thermo Fisher Scientific). Semi-quantitative RT-PCR experi-ments were performed to examine RNA expression of targetgenes. Briefly, 2 lg of total RNA was reverse-transcribed usingSuperScriptVR First-Strand Synthesis System (Thermo FisherScientific). The first-strand cDNA was used as a template. b-Actinwas used as the normalization control.

Neuronal survival assay

The survival assay of amyloid-b neurotoxicity was performed asreported previously.33 At various time periods, neurons were lysed ina cell lysis buffer. Under light microscopy, the numbers of intact nu-clei indicating living cells was quantified and expressed relative to thenumber of cells in the control group without amyloid-b treatment.

Statistical analysis

Unpaired two-tailed t-test and one-way ANOVA with Tukey’s mul-tiple comparison test were performed when groups were com-pared. P-values were calculated using GraphPad Prism softwareversion 8.0. All graphs show means ± standard error of the mean(SEM). Differences were considered significant at *P50.05.

Data availability

Supporting microarray data were downloaded from the Gene ExpressionOmnibus under accession codes GSE15222, GDS2795, GDS810 andGDS4136. All data generated and analysed during this study are availablefrom the corresponding author on reasonable request.

ResultsPAX6 is a downstream target for E2F1/c-Myb

To examine the downstream events in the CDK4/pRB/E2F1 path-way, workflow of data processing is shown in Supplementary Fig.1A and Supplementary material. In an array-based expressionanalysis (GSE15222),22 of the 1389 genes identified as differentiallyexpressed between the 176 Alzheimer’s disease brains and 187healthy brains, 670 genes were predicted to be E2F1 regulatory tar-gets from a ChIP-sequencing dataset. Among them, 16 genes wereconfirmed as brain cell markers25 (Supplementary Fig. 1B). Usingsemantic similarity among three GO terms [biological process (BP),molecular function (MF) and cellular component (CC)] of the 16genes, PAX6 showed the highest overall correlation with E2F1(Supplementary Fig. 1C) and was significantly upregulated inhuman Alzheimer’s disease brains.

Upon further analysis, we identified transcription factor bindingsites in the promoters of several novel downstream targets (e.g.PAX6 and c-Myb) of E2F1. Although PAX6 is a known transcriptionfactor important for eye, brain and olfactory system develop-ment,34–36 there is no information about PAX6 involvement in theE2F1 pathway and its function in Alzheimer’s disease pathogenesis.Both the human and murine Pax6 promoters harboured putativeE2F1 and c-Myb binding sites situated closely together (Fig. 1A andC). We performed ChIP analyses for E2F1 and c-Myb in murine cor-tical neurons and human HEK293 cells and found that boththe mouse and human PAX6 promoters were occupied by E2F1 andc-Myb (Fig. 1B and D). To confirm whether E2F1 and c-Myb transacti-vate PAX6 in vitro, we overexpressed both wild-type E2F1 and c-Mybin murine neuroblastoma cells (N2a) and observed increased PAX6promoter activity in a luciferase promoter assay (Fig. 1E). By con-trast, shRNA-mediated knockdown of E2F1 or c-Myb resulted indecreased PAX6 promoter activity in N2a cells (Fig. 1E). These dataconfirmed that both E2F1 and c-Myb transactivate PAX6.

PAX6 and c-Myb are overexpressed in Alzheimer’sdisease post-mortem brains

We examined the expression profile of PAX6 and c-Myb in an inde-pendent set of human brain tissues (Supplementary Table 3).Western blot of frontal cortical tissue isolated from 14 patientswith Alzheimer’s disease and 14 non-Alzheimer’s disease controls


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showed that expression of PAX6 (Fig. 2A, P = 0.0066) and c-Myb(Fig. 2B, P50.001) was significantly upregulated in Alzheimer’sdisease brains compared with controls (Fig. 2A and B).

PAX6 is increased in the entorhinal cortex of APPtransgenic mice

To determine if PAX6 is also induced in an in vivo Alzheimer’s dis-ease model, we examined whether PAX6 protein is increased inTgCRND8 mice, which overproduce toxic amyloid-b1-42.20 Theentorhinal cortex is the first brain area to be affected inAlzheimer’s disease and its atrophy is highly associated with epi-sodic memory impairment in patients with Alzheimer’s dis-ease.37,38 Immunohistochemical staining of TgCRND8 and wild-type littermate mouse brains showed that beginning at 4 weeks ofage, the proportion of neurons expressing PAX6 was significantlyincreased in the entorhinal cortex of TgCRND8 mice (Fig. 2C). Thisincrease lasted until the mice were 26 weeks old. PAX6 was local-ized exclusively in neurons and inside the nucleus (Fig. 2C).

PAX6 and c-Myb are increased in response toamyloid-b toxicity

To study in detail the molecular mechanism of the CDK/pRB/E2F1pathway, we next examined whether PAX6 or c-Myb expressionchanged in cultured cortical neurons after amyloid-b treatment.Neurons were exposed to oligomeric amyloid-b1-42 (5 lM) for 12, 24or 36 h, and analysed for Pax6 and c-Myb mRNA levels by RT-PCR.We found that the expression of both genes were significantlyincreased after amyloid-b exposure, and both transcripts peaked

following a 12 h treatment (Fig. 3A and E). Consistent with the tran-scriptional upregulation, western blot analysis showed that boththe PAX6 and c-Myb proteins increased in a similar manner (Fig.3B and F). Notably, the basal endogenous mRNA and proteinamounts of both genes were almost undetectable in postmitoticneurons, suggesting that PAX6 and c-Myb might be functionallyquiescent without stress induction.

Next, we investigated whether the upregulation of PAX6 and c-Myb is associated with an increase in transcriptional activation oftheir targets. Cortical neurons were transfected with a P6CON lucifer-ase reporter construct, which contained three standard PAX6 bindingsites39 to measure PAX6 binding; a luciferase construct containingthe PAX6 P1 promoter40 to measure PAX6 promoter activation; orwith a luciferase construct containing c-Myb binding sites to meas-ure c-Myb binding. The same luciferase assays were then performedafter amyloid-b treatment. We found that the amyloid-b challengeinduced PAX6 DNA-binding activity (Fig. 3C), PAX6 promoter activity(Fig. 3D) and c-Myb binding (Fig. 3G). Taken together, these data indi-cate that amyloid-b-induced increase in PAX6 and c-Myb mRNA andprotein levels correlate well with increased transcriptional activity ofPAX6 and c-Myb in Alzheimer’s disease, suggesting that PAX6 and c-Myb might have a role in neuronal apoptosis.

PAX6 and c-Myb are amyloid-b-inducedproapoptotic proteins

We then examined PAX6 and c-Myb function for neuronal apop-tosis. Cortical neurons were transfected with Pax6 or c-Myb siRNAoligonucleotides and treated with amyloid-b1-42 oligomers (5 lM),

Figure 1 E2F1 and c-Myb interact with the PAX6 promoter in both mouse and human cells. (A) Schematic representation of E2F1 binding sites in themouse Pax6 promoter region. (B) ChIP assay showing E2f1 binding to the PAX6 promoter in mouse cortical neurons (left) and HEK293 cells (right). (C)Schematic representation of c-Myb binding sites in the mouse Pax6 promoter region. (D) ChIP assay showing c-Myb binding to the Pax6 promoter inmouse cortical neurons (left) and HEK293 cells (right). (E) PAX6 promoter luciferase assay. A luciferase construct (containing the human wild-typePAX6 promoter) and a Renilla control plasmid were co-transfected into N2a cells with a pcDNA control vector, E2F1 or c-Myb expression plasmid (left)or a shRNA control vector, E2F1 shRNA (middle) or c-Myb shRNA plasmids (right). One day after transfection, cell lysates were processed for both theluciferase and Renilla assay. Data are presented as normalized luciferase activity. Error bars for the figure represent mean ± SEM; *P 5 0.05, ***P 50.001 and ****P 5 0.0001, n = 3; Unpaired two-tailed t-test. Three independent experiments were performed in E, and two in B and D.


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and a survival assay was performed. We found that two Pax6-spe-cific and two c-Myb-specific siRNA oligonucleotides offered signifi-cant protection from amyloid-b1-42 treatment compared with acontrol siRNA (about 40% survival with control siRNA versus 70%with PAX6 knockdown or 60% survival with c-Myb knockdown; Fig.4A). These data suggest that PAX6 and c-Myb are potential media-tors of amyloid-b neurotoxicity.

CDK activity is required for amyloid-b-inducedc-Myb and PAX6 activation

To test the hypothesis that the CDK/pRB/E2F1 pathway acts up-stream of c-Myb and Pax6, we used a potent CDK inhibitor, flavo-piridol. Because multiple CDKs could potentially modulateapoptotic signals, we tested the effect of flavoporidol on neuronal

survival. Flavoporidol protected cortical neurons against amyloid-b-induced death (Fig. 4B) and significantly blocked amyloid-b-induced upregulation of Pax6 and c-Myb mRNA and protein (Fig. 4Cand D). Notably, co-treatment with flavopiridol blocked amyloid-b-induced activation of the Pax6 promoter (Fig. 4E). These findingssuggest that CDK activity is essential for the upregulation and acti-vation of both transcription factors.

E2F1 and c-Myb synergistically transactivate PAX6in amyloid-b-induced toxicity

Next, we addressed the question of whether E2F1 acts upstream ofc-Myb and PAX6 to mediate amyloid-b toxicity. Using siRNA todownregulate E2F1 expression, we found that E2f1 silencing sig-nificantly blocked the upregulation of Pax6 and c-Myb mRNA and

Figure 2 PAX6 expression is significantly increased in the entorhinal cortex of TgCRND8 transgenic mice and Alzheimer’s disease-post mortem brain.(A) PAX6 and (B) c-Myb western blots of frontal cortical brain lysates from normal control (lanes 1–7, 15–21; n = 14) and Alzheimer’s disease tissues(lanes 8–14, 22–28; n = 14). GAPDH was used as a loading control. (C) Brain sections were triple-stained with NeuN antibody (green) anti-Pax6 monoclo-nal antibody (red) and DAPI for 24 h at 4�C. The sections were incubated with Alexa-488-conjugated antibody specific for rabbit IgG and Alexa-594-conju-gated antibody for mouse IgG for 3 h at room temperature. The sections were visualized by confocal microscopy. Scale bar = 20 mm. (D) NeuN-positiveneurons with or without PAX6 staining were counted in layers 2 and 3 of the entorhinal cortex in brains from wild-type and TgCRND8 mice. The countswere obtained by averaging the data from two different experimenters. n = 3 mice per group. Error bars for the figure represent mean ± SEM; *P 5 0.05,**P 5 0.01, ***P 5 0.001 and ****P 5 0.0001; Unpaired two-tailed t-test. Representative images are from two or three independent experiments.


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protein (Fig. 4F and G). Binding of E2F1 to the Pax6 promoter wassignificantly increased after amyloid-b treatment (Fig. 4H). We alsoinvestigated the effect of E2F1 and c-Myb on PAX6 promoter bind-ing and E2F1 on c-Myb promoter binding in post-mortemAlzheimer’s disease brain. ChIP of 10 Alzheimer’s disease and 10control brains showed that all three binding affinities increased inAlzheimer’s disease frontal cortex tissue compared with controlfrontal cortex tissue (Supplementary Fig. 3B–D). Taken together,these findings suggest that E2F1 is responsible for the amyloid-b-induced c-Myb and PAX6 increase.

Furthermore, c-Myb downregulation decreased Pax6 expressionat the mRNA and protein levels (Fig. 4I and J). Additionally, amyl-oid-b treatment of neurons substantially increased occupancy ofthe Pax6 promoter by c-Myb (Fig. 4K). Importantly, this increasecan be blocked by E2f1 silencing (Fig. 4L), supporting an amyloid-b-induced signalling response in which E2F1 can either directly acti-vate PAX6 at the transcriptional level or transactivate PAX6through c-Myb activation.

PAX6 transactivates GSK-3b to promotephosphorylation of tau protein

To identify downstream target genes for Pax6, we performed a com-parative gene expression analysis using RNA sequencing. To thisend, we analysed mRNA samples from murine cortical neurons

transfected with control or Pax6 siRNA. We observed significant dif-ferences in the expression of many Alzheimer’s disease-relatedgenes between the Pax6-silenced and control groups (SupplementaryTable 1). For example, regulator of G-protein signalling 14 (RGS14)was most downregulated; RGS14 is reported to be a key regulator ofsignalling pathways linking synaptic plasticity in CA2 pyramidalneurons to hippocampal-based learning and memory.41 Specifically,we found that PAX6 transcriptionally regulated multiple major kin-ases that phosphorylate tau, including CDK5 and p35, GSK-3b

(encoded by Gsk3b), mitogen-activated protein kinases (MAPK), ser-ine/threonine protein kinase (MARK), calcium/calmodulin-depend-ent protein kinase type II a (CAMK2a) (Supplementary Table 2). Inthis study, we chose to focus on further validating GSK-3b, whichmight be a direct target of PAX6. GSK-3b is a proline-directed serine-threonine kinase that has been postulated to have a role in tau phos-phorylation and neurofibrillary tangle formation.42 In the RNA-sequencing screening for Pax6 target genes, we found that a 40.0%downregulation of Pax6 mRNA causes a 33.5% reduction in GSK-3b

mRNA expression. There were two PAX6 binding sites in the GSK-3b

promoter sequence in both mice and humans.We next examined the PAX6 and GSK-3b interaction by ChIP

assay in untreated HEK293 cells and murine cortical neurons andfound that the GSK-3b promoter region was occupied by PAX6 inboth species (Fig. 5A). Next, we tested the responsiveness of theGSK-3b promoter to PAX6 in an amyloid-b model in mouse

Figure 3 PAX6 and c-Myb are upregulated by amyloid-b in cultured cortical neurons. (A and E) Pax6 and c-Myb mRNA. b-Actin was used as a loadingcontrol. Data for Pax6 or c-Myb expression were normalized to b-actin and to the zero time point, and were compared to untreated controls. (B and F)PAX6 and c-Myb proteins. b-Actin was used as the loading control. (C) PAX6 transcription activity. (D) PAX6 promoter activation by amyloid-b treat-ment. (G) c-Myb transcriptional activity. Error bars for the figure represent mean ± SEM; *P 5 0.05, **P 5 0.01, ***P 5 0.001 and ****P 5 0.0001, n = 3; one-way ANOVA with Tukey’s multiple comparison test. All data are from at least three independent experiments.


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cortical neurons. Amyloid-b treatment significantly increasedPAX6 occupancy of the GSK-3b promoter (Fig. 5B). This increasedbinding affinity was also observed in human Alzheimer’s diseasebrain compared with non-Alzheimer’s disease controls(Supplementary Fig. 3A). Amyloid-b treatment increased GSK-3b

mRNA and protein levels (Fig. 5C and D), which was blocked bysiRNA-mediated PAX6 knockdown (Fig. 5E and F). These data sug-gest that GSK-3b acts as a downstream effector of PAX6 in amyl-oid-b signalling.

Hyperphosphorylation of tau at serine and threonine residuesis a hallmark of neurofibrillary tangles in Alzheimer’s disease.43,44

Since GSK-3b kinase is involved in regulating tau phosphorylation,we reasoned that PAX6 induction might also regulate tau phos-phorylation in our amyloid-b toxicity paradigm. To test this idea,we downregulated PAX6 with siRNA and examined tau phosphor-ylation sites. Amyloid-b challenge increased the concentration ofphospho-tau Ser 356, 396 and 404 (Fig. 5G). When PAX6 was down-regulated, tau phosphorylation at the three serine sites and theratio of phospho-tau to total tau were strongly decreased, suggest-ing that the cell cycle pathway mediates GSK-3b activity in thisparadigm. This result was consistent with a previous report thataggregated amyloid-b can activate GSK-3b to phosphorylate tau.45

Figure 4 CDK/E2F1 regulates PAX6 expression and activity in an amyloid-b model. (A) Downregulation of Pax6 and c-Myb by siRNA protect corticalneurons from death induced by amyloid-b treatment. Cortical neurons were transfected with two Pax6 (left) or two c-Myb (right) siRNA oligonucleoti-des (siPax6-1 or siPax6-2, sic-Myb1 or sic-Myb2) or control siRNA (si-Control) for 24 h and then treated with amyloid-b1-42 for 36 and 48 h. (B) Survivalassay of amyloid-b-induced death of cortical neurons co-treated with flavopiridol. (C) CDK inhibition with flavopiridol decreases amyloid-b-inducedPax6 and c-Myb mRNA upregulation (left). Densitometric analysis of mRNA levels (right). (D) CDK inhibition with flavopiridol decreases amyloid-b-induced upregulation of PAX6, c-Myb and E2F1 protein levels in cortical neurons. b-Actin levels are used as a control for equal input. (E) CDK inhib-ition with flavopiridol decreases amyloid-b-induced activation of the Pax6 promoter. (F) E2f1 knockdown blocks amyloid-b-induced upregulation of c-Myb and Pax6 at the mRNA level. Cortical neurons were transfected with a control siRNA (si-Control) or E2f1 siRNA oligonucleotides (siE2F1) for 24 h,followed by treatment with amyloid-b1-42 for 12 h and 24 h. (G) E2f1 knockdown blocks amyloid-b-induced upregulation of c-Myb and PAX6 at the pro-tein level. b-Actin was used as a loading control. (H) ChIP assay showing that amyloid-b enhances E2F1 occupancy of the Pax6 promoter. (I) RT-PCRand (J) western blot showing siRNA inhibition of c-Myb. b-Actin was used as a loading control. (K) Amyloid-b treatment increases occupancy of thePax6 promoter by c-Myb. (L) E2f1 knockdown decreases occupancy of the Pax6 promoter by c-Myb upon amyloid-b treatment. Error bars for the figurerepresent mean ± SEM; *P 5 0.05, **P 5 0.01, ***P 5 0.001, and ****P 5 0.0001, n = 3; one-way ANOVA with Tukey’s multiple comparison test. All dataare representative of at least three independent experiments.


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DiscussionIn this study, we showed that PAX6 is an important molecularlink between amyloid-b and tau hyperphosphorylation. PAX6 ex-pression is increased in the brains of APP transgenic mice andhuman Alzheimer’s disease patients. Importantly, the in vitro andin vivo findings are corroborated by the observation that expres-sion of PAX6 and E2F1 is increased in the entorhinal cortex neu-rons of patients with mid-stage Alzheimer’s disease, who haveneurofibrillary tangles (GEO Profile Record GDS2795).46 Another re-port suggested that PAX6 expression gradually increases in thehippocampi of patients with Alzheimer’s disease as the diseaseprogresses, with a 1.2-times increase in incipient, 1.3-times in-crease in moderate and 1.8-times increase in severe Alzheimer’sdisease cases.47 c-Myb expression follows the same pattern, with a3.1-times increase for incipient cases, 2.9-times increase for mod-erate cases and 5.1-times increase for severe cases (GEO ProfileRecord GDS810, GDS4136). These findings suggest that PAX6 andc-Myb expression is upregulated throughout the course ofAlzheimer’s disease.

We provide in vitro data supporting a plausible mechanismthrough which amyloid-b activates the cellular signalling path-ways involving CDK, pRB and E2F1 by activating downstream

transcription factors c-Myb and PAX6, upregulating GSK-3b, andultimately leading to the hyperphosphorylation of tau. In this mo-lecular signalling model (Fig. 6), treatment of primary neuronswith amyloid-b activates CDK1 and CDK4/6 death signals, followedby pRB hyperphosphorylation. Subsequently, transcription factorE2F1 is upregulated, turning on transcription and translation ofdownstream target genes Pax6 and c-Myb. c-Myb also transacti-vates PAX6 in response to amyloid-b insults. Subsequently, E2F1and c-Myb synergistically transactivate PAX6. The clinical rele-vance of the amyloid-b-induced upregulation of this pathway iscorroborated by microarray data obtained from post-mortembrains of Alzheimer’s disease patients and a transgenic APP mousemodel. Furthermore, RNAi-mediated downregulation of eitherPAX6 or c-Myb protects cortical neurons from amyloid-b-inducedcell death, indicating that both genes have proapoptotic roles.Importantly, we identified a potential target gene for PAX6, GSK3B(GSK-3b), one of the main kinases that phosphorylates tau protein,possibly leading to the formation of neurofibrillary tangles. Weshowed that amyloid-b neurotoxicity causes PAX6 to transactivateGSK-3b, the upregulation of which was seen in multiple models ofAlzheimer’s disease, as well as in post-mortem humanAlzheimer’s disease brains.48,49 Downregulation of PAX6 blockedthe amyloid-b-induced GSK-3b increase and tau phosphorylation.

Figure 5 PAX6 transactivates GSK-3b and regulates tau phosphorylation in amyloid-b signalling. (A) ChIP assay showing PAX6 binding to the GSK-3bpromoter in mouse cortical neurons (left) and HEK293 cells (right). (B) ChIP assay showing that amyloid-b treatment increases occupancy of the GSK-3b promoter by PAX6 (left) confirmed by densitometric analysis (right). (C) RT-PCR showing increased GSK-3b mRNA and (D) western blot showingincreased GSK-3b protein after amyloid-b treatment. (E) RT-PCR and (F) western blot showing that inhibition of Pax6 by siRNA downregulates amyl-oid-b-induced increase of GSK-3b mRNA and protein. (G) Western blot showing that inhibition of Pax6 by siRNA blocks amyloid-b-induced total tauincrease and Ser356, Ser396 and Ser404 phosphorylation. Relative increase in tau phosphorylation was normalized against tau5. b-Actin was used asa loading control. Error bar for the figure represents mean ± SEM, *P 5 0.05, **P 5 0.01, ***P 5 0.001 and ****P 5 0.0001; P-values were calculated by one-way ANOVA with Tukey’s multiple comparison test in A–G. Data are representative of at least three independent experiments.


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This interaction indicates a potential signalling pathway linkingamyloid-b to tau pathology. However, it is known that inhibitoryserine-phosphorylation also regulates GSK-3b activity. And in thisstudy, the effect of PAX6 on GSK-3b phosphorylation needs to befurther investigated.

Evidence supports the idea that cell cycle re-entry in terminallydifferentiated neurons causes neuronal death.50 However, how cellcycle signals actually trigger neuronal death is largely unknown.E2F1 has been shown to regulate amyloid-b-associated neuronaldeath dependent on a classic mitochondrial pathway includingBcl-2-associated X protein and caspase-3.19 Considering the proa-poptotic role of the transcription factors involved in the identifiedpathway, raises the more specific question of how the E2F1/c-Myb/PAX6 pathway triggers caspase activation. To address this ques-tion, additional downstream targets for these transcription factorsneed to be identified and characterized.

The biological activity of tau is modulated by its phosphoryl-ation status. Although previous studies have shown that post-translational modifications by multiple kinases (GSK-3b, CDK1 and

CDK5, protein kinase A, MAPK) and phosphatases (protein phos-phatase 2A and 2B) contribute to tau hyperphosphorylation andneurofibrillary tangles formation,44,51,52 there is still a missing linkin understanding the mechanism of this regulation. GSK-3b, a ser-ine/threonine, proline-directed kinase is involved in a diversearray of signalling pathways, and strongly implicated inAlzheimer’s disease pathogenesis. GSK-3b can phosphorylate tauat multiple serine residues, and both its protein level and kinaseactivity are increased in Alzheimer’s disease.42,53 Also, GSK-3b

inhibitors have been shown to reduce Alzheimer’s disease path-ology in vivo and in preclinical trials.54,55 Importantly, our datashowed a novel pathway for hyperphosphorylation of tau protein.Although we focused on the hyperphosphorylation of tau proteinin this study, we also observed that total tau was reduced afterPax6 knockdown in RNA and protein level (Supplementary Table 2and Fig. 5G). Whether PAX6 regulates total tau directly or indirectlyneeds further study.

When first identified as Alzheimer’s disease-associated factors,the cause-effect relationship between amyloid-b and tau was not

Figure 6 Proposed model for the role of PAX6 in amyloid-b induced neurotoxicity.


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clear, but extensive studies have been performed in that regard.For example, Amar et al.56 show that amyloid-b evoked an increasein tau phosphorylation dependent on CaMKIIa activation. Thisinteraction is supported by our RNA-sequencing data and seems toalso involve Pax6 (Supplementary Table 1). Additionally, our RNA-sequencing data (Supplementary Table 2) indicated that Pax6

silencing in neurons could significantly decrease the expressionlevels of several key kinases that phosphorylate tau, such as Cdk5

(17.3%) and Mapk1 (63.8%). Therefore, there are probably more tar-gets and downstream pathways of PAX6 involved in Alzheimer’sdisease pathogenesis.

In summary, our study provides evidence that amyloid-bneurotoxicity leads to hyperphosphorylation of tau through acti-vation of signalling cascades normally associated with cell-cycleactivation in neurons, subsequent activation of transcription fac-tors such as c-Myb and PAX6 and hyperactivation of GSK-3b. Thebasal endogenous mRNA and protein levels of both c-Myb or PAX6are almost undetectable in post-mitotic neurons, suggesting thatPAX6 and c-Myb might be functionally quiescent without stress in-duction, but are upregulated significantly in Alzheimer’s diseasebrains. Moreover, neurons are dying at all stages of Alzheimer’sdisease, even if amyloid-b is totally blocked. Therefore, a plausibletherapeutic strategy is to combine amyloid-b removal and target-ing PAX6 to prevent neuronal death and tau hyperphosphoryla-tion, therefore to slow Alzheimer’s disease progression. Thepathway that we identified suggests E2F1, c-Myb or PAX6 as noveltargets for pharmaceutical intervention.

AcknowledgementWe thank Changyuan Song for lifelong support for research inAlzheimer’s disease.

FundingThis work was supported by grants from the National NaturalScience Foundation of China (No. 81271226), the seed funding ofthe University of Hong Kong, and the Research Grant council of theHong Kong Special Administrative Region (No. 11200218). E.F.F.was supported by HELSE SØR-ØST (#2017056, #2020001, #2021021),the Research Council of Norway (#262175 and #277813), theNational Natural Science Foundation of China (#81971327), and anAkershus University Hospital Strategic grant (#269901).

Competing interestsE.F.F. has CRADA arrangements with ChromaDex and is a consult-ant to Aladdin Healthcare Technologies and the VancouverDementia Prevention Centre.

Supplementary materialSupplementary material is available at Brain online.

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amyloid-b toxicity modulates tau phosphorylation through

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