doi:10.

1093/brain/awab134 BRAIN 2021: 144; 2759–2770 | 2759

Amyloid-b toxicity modulates tau

phosphorylation through the PAX6
signalling pathway

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Yalun 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 of
Alzheimer’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 tau
protein. 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 in
adults, is increased in the brains of patients with Alzheimer’s disease and in APP transgenic mice, and plays a key
role between amyloid-b and tau hyperphosphorylation.
Downregulation of PAX6 protects against amyloid-b peptide-induced neuronal death, suggesting that PAX6 is a
key 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 and
neurofibrillary 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 by
activating 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, China

2 HKU-Shenzhen Institute of Research and Innovation, University of Hong Kong, Hong Kong, China
3 Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON, M5T 0S8, Canada
4 Department of Medical Biophysics, and Medicine (Neurology), University of Toronto, Krembil Discovery Tower,
Toronto, ON, M5T 2S8, Canada
5 Department of Clinical Molecular Biology, University of Oslo and the Akershus University Hospital, 1478 Lørenskog,
Norway
6 School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China
7 Department of Ophthalmology, University of Hong Kong, Hong Kong, China
8 Department of Genetics, National Research Institute for Family Planning, Beijing, China
9 Cambridge Institute for Medical Research, Department of Clinical Neurosciences, University of Cambridge,
Cambridge CB2 0XY, UK
10 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, 2021
C The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
V
For permissions, please email: [email protected]
2760 | BRAIN 2021: 144; 2759–2770 Y. Zhang et al.

Correspondence to: Youqiang Song

School of Biomedical Sciences
University of Hong Kong, Hong Kong, China
E-mail: [email protected]

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

Abbreviation: ChIP = chromatin immunoprecipitation

Introduction used. For chromatin immunoprecipitation, anti-Pax6 (Santa Cruz,

sc-32766, 1:50), anti-c-Myb (Santa Cruz, sc-74512, 1:50), anti-E2F1
Genetic and cell biology studies have shown an important role for (Santa Cruz, sc-251, 1:50), and anti-Flag (Sigma, F1804, 1:500) were

Downloaded from https://academic.oup.com/brain/article/144/9/2759/6356359 by guest on 25 March 2023

amyloid-b peptide and microtubule-associated protein tau in the used. For immunostaining, anti-Pax6 (Covance, PRB-278P, 1:2000),
pathogenesis of Alzheimer’s disease.1,2 The molecular pathways anti-NeuN (Chemicon, MAB377, 1:1000), secondary antibodies goat
linking amyloid-b, tau and cell death are controversial.3–5 Some evi- R
anti-rabbit Alexa FluorV 568 (Thermo Fisher Scientific, A-11011,
dence from experiments, including in vitro studies of neuronal cell R
1:4000) and goat anti-mouse Alexa FluorV 488 (Thermo Fisher
death, in vivo investigation of animal models, and human post-mor- Scientific, A-11008, 1:4000) were used. For primary neuron treat-
tem studies, support a hypothesis that implicates deregulation of ment, amyloid-b1-42 peptide was purchased from Bachem (H1368),
cell cycle proteins as key mediators of neuronal dysfunction and and flavopiridol was purchased from Sigma (F3055).
loss in Alzheimer’s disease brains.6,7 For example, cell division cycle
25 (CDC25) phosphatases have increased expression and activity in
Plasmid constructs
Alzheimer’s disease brains.8,9 Multiple cyclin-dependent kinases
(CDKs: CDK1, CDK4 and CDK6), retinoblastoma protein (pRB), cyclins PAX6 P1 promoter luciferase reporter construct pGL3b-Pax6 (1–346)
(A, B, C, D and E) and CDK inhibitors are overexpressed in cellular was a gift from Dr Andrew Chantry; PAX6 luciferase reporter con-
and animal models and post-mortem brains of patients with struct P6CON-luc, PQ107 was a gift from Dr Ales Cvekl; c-Myb shRNA
Alzheimer’s disease.10–19 Central to the hypothesis of the involve- construct pSiren-retroQ-Myb and control construct pSiren-retroQ-
ment of cell cycle deregulation in neuronal death in Alzheimer’s Luc were gifts from Dr Robert K. Slany; c-Myb responsive reporter
disease is the CDK/pRB/E2F1 pathway, wherein amyloid-b activates construct p5xMRE-A-luc (5xMRE) and wild-type c-Myb construct
CDK4/6, leading to pRB phosphorylation and activation of E2F1 tran- pcDNA3.1-FL-c-Myb were gifts from Dr Juraj Bies; E2F1 shRNA con-
scription factor. However, the precise detail of the mechanism by struct BS/U6 E2F1 RNAi and control construct BS/U6 RNAi vector
which amyloid-b accumulation is linked to neurofibrillary tangles were gifts from Dr W. Douglas Cress; and wild-type E2F1 construct
pathology in Alzheimer’s disease remains elusive. pcDNA3.1-HA-E2F1 was a gift from Dr Joseph R. Nevins.

Chromatin immunoprecipitation
Materials and methods
Chromatin immunoprecipitation (ChIP) experiments were con-
Human brain specimens and animals ducted as previously described.21 Mouse cortical neurons and
Post-mortem human brain tissues from the frontal cortex of HEK293 cells were harvested and cell lysates were sonicated and
patients with Alzheimer’s disease and non-demented control sub- centrifuged, and the supernatants collected for immunoprecipita-
jects were obtained from the Canadian Brain Tissue Bank and New tion. Brain samples from 10 Alzheimer’s disease cases and 10 con-
York Brain Bank of Columbia University. The study of human brain trols were obtained from the University of Columbia. Equal
tissue was approved by the Ethics Committee of the Faculty of amounts (30–40 mg) of frozen tissue from each sample were
Medicine of the University of Toronto (N0: 00026798) and the weighed and thawed on ice. Tissues were chopped with a scalpel
University of Hong Kong (N0: UW 13–177). All mice were on a and transferred to conical tubes with ice-cold PBS containing pro-
C57BL/6 genetic background. TgCRND8 mice express human amyl- tease inhibitor cocktail (Sigma). Tissues were cross-linked with 1%
oid precursor protein 695 (APP695) with the Swedish mutation formaldehyde in PBS for 10 min at room temperature and 125 mM
(KM670/671NL) and Indiana mutation (V717F) under the control of glycine was added to quench the reaction. Tissues were centri-
the Prpn (PrP) gene promoter.20 All experiments using animals fuged at low speed (1300 rpm) at 4 C for 5 min and the supernatant
were approved by the Committee for the Use of Live Animals in was removed. The pellets were washed with ice-cold PBS twice,
Teaching and Research at the University of Hong Kong (N0: centrifuged and resuspended in lysis buffer (1% SDS, 10 mM EDTA,
CULATR2792-12, CULATR 3732-15, CULATR 5212-19). 50 mM Tris/HCl, pH 8.1) containing protease inhibitor cocktail
(Sigma). Next, pellets were homogenized with a Dounce tissue
grinder pestle in 20 strokes, and lysates were sheared by sonic-
Antibodies and reagents ation for 50–100 s in 10 s bursts. Samples were centrifuged at 4 C
For western blot, anti-Pax6 (Thermo Fisher Scientific, 42-6600, for 10 min at 15 000 rpm to remove debris, and supernatants were
1:1000), anti-Pax6 (Sigma, SAB5300039, 1:1000), anti-c-Myb (Cell diluted 1:10 in a lysis buffer (0.01% SDS, 1% TritonTM X-100, 2 mM
Signaling, 12319, 1:1000), anti-c-Myb (Abcam, ab117635, 1:3000), anti- EDTA, 20 mM Tris/HCl, pH 8.1, 150 mM NaCl). An aliquot of 20 ml
E2F1 (Santa Cruz, sc-251, 1:1000), anti-GSK-3b (Cell Signaling, 9315, from each undiluted sample was stored for the input chromatin.
1:3000), anti-phospho-Tau (pSer356) (Sigma, SAB4504556, 1:1000), Immunoprecipitations were performed with specific antibodies at
anti-phospho-Tau (pSer396) (Sigma, T7319, 1:3000), anti-phospho- 4 C overnight with rotation, a non-specific anti-Flag antibody was
Tau (pSer404) (Sigma, T7444, 1:3000), anti-b-actin (Cell Signaling, used as a negative control. ChIP-Grade Protein G Magnetic Beads
4970, 1:3000), and anti-GAPDH (Cell Signaling, 2118, 1:3000) were slurry (30 ll) (#9006, Cell Signaling) was added to each sample and
PAX6 links between amyloid-b and p-tau BRAIN 2021: 144; 2759–2770 | 2761

then incubated with rotation for 2 h. Beads were pelleted using a Aligned reads summarization and transcriptome
magnetic separation rack and washed four times with washing reconstruction using Cufflinks
buffer. Beads were eluted twice with elution buffer (1% SDS, 0.1 M
Aligned RNA read files in BAM format were inputted into Cufflinks
NaHCO3) by rotation at 30 C for 15 min. Formaldehyde cross-link-
(version 2.0.0) to assemble the aligned reads into transcripts.
ing was reversed by overnight heating at 65 C, as well as input,
Cufflinks uses a reference genome-guided method to assemble
incubating for 1 h with RNase A at 37 C (20 mg/ml) and then for 1 h
exons, identify novel transcripts and report a minimal set of iso-
with proteinase K at 42 C (20 mg/ml). DNA was purified using a PCR
forms to best describe the reads in the dataset.28 Cufflinks normal-
purification kit (Qiagen), and samples were analysed by semi-
izes the raw fragment counts with a maximum likelihood
quantitative PCR.
estimation method and quantifies the expression of transcripts as
a fragments per kilobase of exon model per million mapped frag-
Mining human brain microarray datasets ments (FPKM) value. Cuffmerge was used to merge all transcript
The workflow of data processing is shown in Supplementary Fig. assemblies in GTF format to construct a more complete and accur-
1A. Four steps were performed to screen and rank genes regulated ate transcriptome. This transcriptome was then compared with
by E2F1. First, gene expression profile data for GSE1522222 were the mouse reference annotation file downloaded from Ensembl

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obtained from the Gene Expression Omnibus (GEO) database (plat- (NCBIM37) to identify novel genes and transcripts by
form GPL2700). Brain samples with a confirmed pathological diag- Cuffcompare.29
nosis of late-onset Alzheimer’s disease (n = 176) and control brains
(n = 187) were extracted. Gene differential expression was assessed Differential expression test using Cuffdiff and
by R package Limma (version 3.40.6),23 and genes with fold change candidate gene selection
41.5 and adjusted P-value 50.05 were categorized as differentially
Cuffdiff, which is included in the Cufflinks package, was used to
expressed genes. ChIP-sequencing datasets for human transcrip-
detect differentially expressed genes or transcripts between the
tion factor binding sites were downloaded for annotation and
normal control and the Pax6 knockdown groups in the mouse
screening of the downstream targets of E2F1.24 Potential E2F1
RNA-sequencing experiment. Differential gene expression after
downstream regulatory genes were derived by overlapping E2F1
Pax6 knockdown was calculated by the ratio of FPKM value at P0
transcription factor binding sites and the promoter regions (2 kb
versus C0. In total, 8584 genes (4520 downregulated and 4064 upre-
upstream to 1 kb downstream of the transcription start site for
gulated) were identified as differentially expressed with false dis-
each gene) of the differentially expressed genes. Gene markers in
covery rate-adjusted P-values 5 0.05. Pathway enrichment
the human brain were extracted from the Cell Marker database25
analysis based on these differentially expressed genes identified
for further screening. Finally, Gene Ontology (GO) semantic simi-
60 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG)
larity scores of the selected genes were assessed by R package
pathways with P-values 5 10–4. Alzheimer’s disease-related KEGG
GOSemSim (version 2.10.0).26 The overall relevance between E2F1 pathways were defined as pathways that share at least one gene
and the selected genes was calculated based on the arithmetic with the Alzheimer’s disease pathway hsa05010; 77 Alzheimer’s
average value of the three GO semantic similarity scores (biological disease-related KEGG pathways were identified. As a result, 34
process, molecular function and cellular component). Alzheimer’s disease-related enriched KEGG pathways were chosen
and genes involved in these pathways were downloaded from the
RNA-sequencing KEGG database.30 We searched the promoter regions of all differ-
entially expressed genes for PAX6 binding sites using a PAX6 bind-
Total mRNA was extracted from cultured mouse primary neurons
ing matrix from JASPAR. Finally, 33 genes were selected that met
transfected with a control siRNA and Pax6 siRNA. For each control
all of the following criteria: 41.2 expression fold change, predicted
siRNA or Pax6 siRNA treatment, one biological replicate was pre-
to have PAX6 binding sites and involved in at least three
pared. All samples were sequenced by Axeq using Illumina HiSeq
Alzheimer’s disease-related enriched KEGG pathways
2x100 bp paired-end sequencing. Eight raw sequence files were
(Supplementary Table 1).
generated in FASTQ format. The quality of RNA-sequencing raw
reads was evaluated using NGS QC Toolkit (version 2.2.3). The total
number of reads per sequence file was around 32 million and the Primary neuronal cultures and RNA interference
average read length was 101 bp. The overall PHRED quality scores Primary cortical neurons were derived from embryonic Day 14.5
for all sequence files was higher than 20 and the peak value of GC (E14.5) C57BL/6 mice.31 The brains were dissociated from skulls,
content distribution for each sequence file was 45–55%. No further meninges removed and cortices isolated. Individual cells were dis-
trimming or filtering was applied to the raw data. sociated by incubation with trypsin and DNase (Sigma). Cells were
plated on poly-D-lysine (Sigma) coated 24-well plates at a density
Mapping RNA-sequencing reads to a mouse of 1.5  106 cells/ml in NeurobasalTM medium (Thermo Fisher
Scientific) supplemented with B-27 (Thermo Fisher Scientific), N-2
reference genome using TopHat
(Thermo Fisher Scientific), and glutamine (0.5 mM, Thermo Fisher
Millions of short reads from RNA-sequencing were aligned to the Scientific). Three days after seeding, cultured cells were trans-
mouse reference genome using TopHat (version 2.0.3). TopHat first V
fected with siRNAs (60 pmol/24-well) using Lipofectamine 2000
R

applies an unspliced aligner, Bowtie, to align exon reads to a refer- transfection reagent (Thermo Fisher Scientific). Twenty-four hours
ence genome without considering any large gaps and then deals after transfection, cells were exposed to amyloid-b1-42 (5 mM) for
with the small portion of unmapped reads at splicing junctions.27 the indicated time periods and then processed for subsequent
The mouse reference genome sequence was downloaded from the assays. SiRNAs from Santa Cruz are provided as pools consisting
TopHat website (Mus musculus iGenome Ensembl NCBIM37). of three to five target-specific 19–25 nucleotides siRNAs designed
TopHat accepts raw reads files in FASTQ format as input and out- to specifically knockdown the expression of mouse PAX6, c-Myb or
R
puts the alignment results in BAM format. Default parameters E2F1. SilencerV Select pre-designed siRNAs were from Ambion,
were used to run TopHat. Over 80% of the reads could be mapped non-targeting siRNA was used as a negative control. Knockdown
to the reference genome. efficiency per target was tested by RT-PCR or western blot.
2762 | BRAIN 2021: 144; 2759–2770 Y. Zhang et al.

Luciferase reporter assay Neuronal survival assay

Cortical neurons were transfected with luciferase reporter The survival assay of amyloid-b neurotoxicity was performed as
constructs three days after plating. After amyloid-b treatment, reported previously.33 At various time periods, neurons were lysed in
luciferase activity of a target gene was determined using the dual- a cell lysis buffer. Under light microscopy, the numbers of intact nu-
luciferase reporter assay system (Promega) in a microplate lumin- clei indicating living cells was quantified and expressed relative to the
ometer. Luciferase activity was normalized against Renilla activity number of cells in the control group without amyloid-b treatment.
for the transfection efficiency using a pRL Renilla luciferase control
reporter construct. Statistical analysis
Unpaired two-tailed t-test and one-way ANOVA with Tukey’s mul-
Western blot analysis tiple comparison test were performed when groups were com-
pared. P-values were calculated using GraphPad Prism software
Human brain tissues or mouse cortical neurons were dissolved in
version 8.0. All graphs show means ± standard error of the mean
ice-cold 1  RIPA buffer (Cell Signaling) with proteinase inhibitor (SEM). Differences were considered significant at *P 5 0.05.
cocktail (Sigma). Proteins were separated on NuPAGETM Novex

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4–12% Bis-Tris gels (Thermo Fisher Scientific) and transferred to
Data availability
nitrocellulose membranes. Membranes were probed with indi-
cated primary antibodies. The blots were analysed and quantified Supporting microarray data were downloaded from the Gene Expression
by densitometry using ImageJ software (Version 1.52t, National Omnibus under accession codes GSE15222, GDS2795, GDS810 and
Institutes of Health). GDS4136. All data generated and analysed during this study are available
from the corresponding author on reasonable request.

Immunostaining
Results
TgCRND8 mice and non-transgenic littermate pairs at 2, 4, 6, 8, 13,
26 weeks of age were sacrificed by transcardial perfusion with 1 PAX6 is a downstream target for E2F1/c-Myb
PBS, brains were fixed in 4% paraformaldehyde and cryoprotected.
To examine the downstream events in the CDK4/pRB/E2F1 path-
Horizontal sections were cryostat-cut at 10 mm thickness from OCT
way, workflow of data processing is shown in Supplementary Fig.
embedded frozen blocks and mounted onto gelatin-coated slides.
1A and Supplementary material. In an array-based expression
The sections were blocked with 10% goat serum (Sigma) in antibody analysis (GSE15222),22 of the 1389 genes identified as differentially
diluent (Dako) for 1 h at room temperature. The sections were incu- expressed between the 176 Alzheimer’s disease brains and 187
bated overnight at 4 C with: Rabbit anti-Pax6 (Covance, 1:2000) and healthy brains, 670 genes were predicted to be E2F1 regulatory tar-
mouse anti-NeuN (Chemicon, 1:1000) antibodies [prepared in anti- gets from a ChIP-sequencing dataset. Among them, 16 genes were
body diluent (Dako)], followed by incubation with second antibodies confirmed as brain cell markers25 (Supplementary Fig. 1B). Using
R
goat anti-mouse antibodies (Alexa FluorV 488, Thermo Fisher semantic similarity among three GO terms [biological process (BP),
R
Scientific, 1:4000) or goat anti-rabbit antibodies (Alexa FluorV 568, molecular function (MF) and cellular component (CC)] of the 16
Thermo Fisher Scientific, 1:4000) for 1 h. The slides were mounted genes, PAX6 showed the highest overall correlation with E2F1
in a DAPI/Antifade mounting medium (Vectashield). Fluorescent (Supplementary Fig. 1C) and was significantly upregulated in
images were captured using a confocal laser-scanning microscope human Alzheimer’s disease brains.
(LSM 710, Carl Zeiss). Upon further analysis, we identified transcription factor binding
sites in the promoters of several novel downstream targets (e.g.
PAX6 and c-Myb) of E2F1. Although PAX6 is a known transcription
Amyloid-b preparation factor important for eye, brain and olfactory system develop-
Oligomeric amyloid-b1-42 peptide (Bachem) was prepared as ment,34–36 there is no information about PAX6 involvement in the
described previously.32 Briefly, lyophilized, HPLC-purified amyloid- E2F1 pathway and its function in Alzheimer’s disease pathogenesis.
b1-42 was equilibrated and reconstituted in 100% 1,1,1,3,3,3 hexa- Both the human and murine Pax6 promoters harboured putative
fluoro-2-propanol (HFIP; Sigma) to 1 mM. HFIP was evaporated and E2F1 and c-Myb binding sites situated closely together (Fig. 1A and
the crystallized peptide was air-dried and was reconstituted to C). We performed ChIP analyses for E2F1 and c-Myb in murine cor-
5 mM in dimethyl sulphoxide (DMSO) followed by sonication. The tical neurons and human HEK293 cells and found that both
the mouse and human PAX6 promoters were occupied by E2F1 and
5 mM amyloid-b1-42 stock solution was then diluted with phos-
c-Myb (Fig. 1B and D). To confirm whether E2F1 and c-Myb transacti-
phate-buffered saline (PBS) to 400 mM and was incubated for 18–
vate PAX6 in vitro, we overexpressed both wild-type E2F1 and c-Myb
24 h at 37 C. The solution was diluted again for a working concen-
in murine neuroblastoma cells (N2a) and observed increased PAX6
tration of 100 lM. The resulting solution was further incubated at
promoter activity in a luciferase promoter assay (Fig. 1E). By con-
37 C for 18–24 h and the amyloid-b peptide was ready for use.
trast, shRNA-mediated knockdown of E2F1 or c-Myb resulted in
decreased PAX6 promoter activity in N2a cells (Fig. 1E). These data
Semi-quantitative RT-PCR confirmed that both E2F1 and c-Myb transactivate PAX6.
R
Total mRNA was prepared from cell cultures utilizing TRIzolV re-
agent (Thermo Fisher Scientific). Semi-quantitative RT-PCR experi-
PAX6 and c-Myb are overexpressed in Alzheimer’s
ments were performed to examine RNA expression of target disease post-mortem brains
genes. Briefly, 2 lg of total RNA was reverse-transcribed using We examined the expression profile of PAX6 and c-Myb in an inde-
R
SuperScriptV First-Strand Synthesis System (Thermo Fisher pendent set of human brain tissues (Supplementary Table 3).
Scientific). The first-strand cDNA was used as a template. b-Actin Western blot of frontal cortical tissue isolated from 14 patients
was used as the normalization control. with Alzheimer’s disease and 14 non-Alzheimer’s disease controls
PAX6 links between amyloid-b and p-tau BRAIN 2021: 144; 2759–2770 | 2763

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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 the
mouse 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 in
mouse cortical neurons (left) and HEK293 cells (right). (E) PAX6 promoter luciferase assay. A luciferase construct (containing the human wild-type
PAX6 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 the
luciferase and Renilla assay. Data are presented as normalized luciferase activity. Error bars for the figure represent mean ± SEM; *P 5 0.05, ***P 5
0.001 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.

showed that expression of PAX6 (Fig. 2A, P = 0.0066) and c-Myb following a 12 h treatment (Fig. 3A and E). Consistent with the tran-
(Fig. 2B, P 5 0.001) was significantly upregulated in Alzheimer’s scriptional upregulation, western blot analysis showed that both
disease brains compared with controls (Fig. 2A and B). the PAX6 and c-Myb proteins increased in a similar manner (Fig.
3B and F). Notably, the basal endogenous mRNA and protein
PAX6 is increased in the entorhinal cortex of APP amounts of both genes were almost undetectable in postmitotic
transgenic mice neurons, suggesting that PAX6 and c-Myb might be functionally
quiescent without stress induction.
To determine if PAX6 is also induced in an in vivo Alzheimer’s dis- Next, we investigated whether the upregulation of PAX6 and c-
ease model, we examined whether PAX6 protein is increased in
Myb is associated with an increase in transcriptional activation of
TgCRND8 mice, which overproduce toxic amyloid-b1-42.20 The
their targets. Cortical neurons were transfected with a P6CON lucifer-
entorhinal cortex is the first brain area to be affected in
ase reporter construct, which contained three standard PAX6 binding
Alzheimer’s disease and its atrophy is highly associated with epi-
sites39 to measure PAX6 binding; a luciferase construct containing
sodic memory impairment in patients with Alzheimer’s dis-
the PAX6 P1 promoter40 to measure PAX6 promoter activation; or
ease.37,38 Immunohistochemical staining of TgCRND8 and wild-
with a luciferase construct containing c-Myb binding sites to meas-
type littermate mouse brains showed that beginning at 4 weeks of
ure c-Myb binding. The same luciferase assays were then performed
age, the proportion of neurons expressing PAX6 was significantly
increased in the entorhinal cortex of TgCRND8 mice (Fig. 2C). This after amyloid-b treatment. We found that the amyloid-b challenge
increase lasted until the mice were 26 weeks old. PAX6 was local- induced PAX6 DNA-binding activity (Fig. 3C), PAX6 promoter activity
ized exclusively in neurons and inside the nucleus (Fig. 2C). (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 and
protein levels correlate well with increased transcriptional activity of
PAX6 and c-Myb are increased in response to
PAX6 and c-Myb in Alzheimer’s disease, suggesting that PAX6 and c-
amyloid-b toxicity Myb might have a role in neuronal apoptosis.
To study in detail the molecular mechanism of the CDK/pRB/E2F1
pathway, we next examined whether PAX6 or c-Myb expression PAX6 and c-Myb are amyloid-b-induced
changed in cultured cortical neurons after amyloid-b treatment.
proapoptotic proteins
Neurons were exposed to oligomeric amyloid-b1-42 (5 lM) for 12, 24
or 36 h, and analysed for Pax6 and c-Myb mRNA levels by RT-PCR. We then examined PAX6 and c-Myb function for neuronal apop-
We found that the expression of both genes were significantly tosis. Cortical neurons were transfected with Pax6 or c-Myb siRNA
increased after amyloid-b exposure, and both transcripts peaked oligonucleotides and treated with amyloid-b1-42 oligomers (5 lM),
2764 | BRAIN 2021: 144; 2759–2770 Y. Zhang et al.

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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-positive
neurons 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 counts
were 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.

and a survival assay was performed. We found that two Pax6-spe- survival. Flavoporidol protected cortical neurons against amyloid-
cific and two c-Myb-specific siRNA oligonucleotides offered signifi- b-induced death (Fig. 4B) and significantly blocked amyloid-b-
cant protection from amyloid-b1-42 treatment compared with a induced upregulation of Pax6 and c-Myb mRNA and protein (Fig. 4C
control siRNA (about 40% survival with control siRNA versus 70% and D). Notably, co-treatment with flavopiridol blocked amyloid-b-
with PAX6 knockdown or 60% survival with c-Myb knockdown; Fig. induced activation of the Pax6 promoter (Fig. 4E). These findings
4A). These data suggest that PAX6 and c-Myb are potential media- suggest that CDK activity is essential for the upregulation and acti-
tors of amyloid-b neurotoxicity. vation of both transcription factors.

CDK activity is required for amyloid-b-induced E2F1 and c-Myb synergistically transactivate PAX6
c-Myb and PAX6 activation in amyloid-b-induced toxicity
To test the hypothesis that the CDK/pRB/E2F1 pathway acts up- Next, we addressed the question of whether E2F1 acts upstream of
stream of c-Myb and Pax6, we used a potent CDK inhibitor, flavo- c-Myb and PAX6 to mediate amyloid-b toxicity. Using siRNA to
piridol. Because multiple CDKs could potentially modulate downregulate E2F1 expression, we found that E2f1 silencing sig-
apoptotic signals, we tested the effect of flavoporidol on neuronal nificantly blocked the upregulation of Pax6 and c-Myb mRNA and
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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 loading
control. 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.

protein (Fig. 4F and G). Binding of E2F1 to the Pax6 promoter was transfected with control or Pax6 siRNA. We observed significant dif-
significantly increased after amyloid-b treatment (Fig. 4H). We also ferences in the expression of many Alzheimer’s disease-related
investigated the effect of E2F1 and c-Myb on PAX6 promoter bind- genes between the Pax6-silenced and control groups (Supplementary
ing and E2F1 on c-Myb promoter binding in post-mortem Table 1). For example, regulator of G-protein signalling 14 (RGS14)
Alzheimer’s disease brain. ChIP of 10 Alzheimer’s disease and 10 was most downregulated; RGS14 is reported to be a key regulator of
control brains showed that all three binding affinities increased in signalling pathways linking synaptic plasticity in CA2 pyramidal
Alzheimer’s disease frontal cortex tissue compared with control neurons to hippocampal-based learning and memory.41 Specifically,
frontal cortex tissue (Supplementary Fig. 3B–D). Taken together, we found that PAX6 transcriptionally regulated multiple major kin-
these findings suggest that E2F1 is responsible for the amyloid-b- ases that phosphorylate tau, including CDK5 and p35, GSK-3b
induced c-Myb and PAX6 increase. (encoded by Gsk3b), mitogen-activated protein kinases (MAPK), ser-
Furthermore, c-Myb downregulation decreased Pax6 expression ine/threonine protein kinase (MARK), calcium/calmodulin-depend-
at the mRNA and protein levels (Fig. 4I and J). Additionally, amyl- ent protein kinase type II a (CAMK2a) (Supplementary Table 2). In
oid-b treatment of neurons substantially increased occupancy of this study, we chose to focus on further validating GSK-3b, which
the Pax6 promoter by c-Myb (Fig. 4K). Importantly, this increase might be a direct target of PAX6. GSK-3b is a proline-directed serine-
can be blocked by E2f1 silencing (Fig. 4L), supporting an amyloid-b- threonine kinase that has been postulated to have a role in tau phos-
induced signalling response in which E2F1 can either directly acti- phorylation and neurofibrillary tangle formation.42 In the RNA-
vate PAX6 at the transcriptional level or transactivate PAX6 sequencing screening for Pax6 target genes, we found that a 40.0%
through c-Myb activation. 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.
PAX6 transactivates GSK-3b to promote
We next examined the PAX6 and GSK-3b interaction by ChIP
phosphorylation of tau protein assay in untreated HEK293 cells and murine cortical neurons and
To identify downstream target genes for Pax6, we performed a com- found that the GSK-3b promoter region was occupied by PAX6 in
parative gene expression analysis using RNA sequencing. To this both species (Fig. 5A). Next, we tested the responsiveness of the
end, we analysed mRNA samples from murine cortical neurons GSK-3b promoter to PAX6 in an amyloid-b model in mouse
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Figure 4 CDK/E2F1 regulates PAX6 expression and activity in an amyloid-b model. (A) Downregulation of Pax6 and c-Myb by siRNA protect cortical
neurons 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) Survival
assay of amyloid-b-induced death of cortical neurons co-treated with flavopiridol. (C) CDK inhibition with flavopiridol decreases amyloid-b-induced
Pax6 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-PCR
and (J) western blot showing siRNA inhibition of c-Myb. b-Actin was used as a loading control. (K) Amyloid-b treatment increases occupancy of the
Pax6 promoter by c-Myb. (L) E2f1 knockdown decreases occupancy of the Pax6 promoter by c-Myb upon amyloid-b treatment. 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 representative of at least three independent experiments.

cortical neurons. Amyloid-b treatment significantly increased Since GSK-3b kinase is involved in regulating tau phosphorylation,
PAX6 occupancy of the GSK-3b promoter (Fig. 5B). This increased we reasoned that PAX6 induction might also regulate tau phos-
binding affinity was also observed in human Alzheimer’s disease phorylation in our amyloid-b toxicity paradigm. To test this idea,
brain compared with non-Alzheimer’s disease controls we downregulated PAX6 with siRNA and examined tau phosphor-
(Supplementary Fig. 3A). Amyloid-b treatment increased GSK-3b ylation sites. Amyloid-b challenge increased the concentration of
mRNA and protein levels (Fig. 5C and D), which was blocked by phospho-tau Ser 356, 396 and 404 (Fig. 5G). When PAX6 was down-
siRNA-mediated PAX6 knockdown (Fig. 5E and F). These data sug- regulated, tau phosphorylation at the three serine sites and the
gest that GSK-3b acts as a downstream effector of PAX6 in amyl- ratio of phospho-tau to total tau were strongly decreased, suggest-
oid-b signalling. ing that the cell cycle pathway mediates GSK-3b activity in this
Hyperphosphorylation of tau at serine and threonine residues paradigm. This result was consistent with a previous report that
is a hallmark of neurofibrillary tangles in Alzheimer’s disease.43,44 aggregated amyloid-b can activate GSK-3b to phosphorylate tau.45
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Figure 5 PAX6 transactivates GSK-3b and regulates tau phosphorylation in amyloid-b signalling. (A) ChIP assay showing PAX6 binding to the GSK-3b
promoter 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 showing
increased 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 tau
increase and Ser356, Ser396 and Ser404 phosphorylation. Relative increase in tau phosphorylation was normalized against tau5. b-Actin was used as
a 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.

Discussion transcription factors c-Myb and PAX6, upregulating GSK-3b, and

ultimately leading to the hyperphosphorylation of tau. In this mo-
In this study, we showed that PAX6 is an important molecular lecular signalling model (Fig. 6), treatment of primary neurons
link between amyloid-b and tau hyperphosphorylation. PAX6 ex- with amyloid-b activates CDK1 and CDK4/6 death signals, followed
pression is increased in the brains of APP transgenic mice and by pRB hyperphosphorylation. Subsequently, transcription factor
human Alzheimer’s disease patients. Importantly, the in vitro and E2F1 is upregulated, turning on transcription and translation of
in vivo findings are corroborated by the observation that expres- downstream target genes Pax6 and c-Myb. c-Myb also transacti-
sion of PAX6 and E2F1 is increased in the entorhinal cortex neu- vates PAX6 in response to amyloid-b insults. Subsequently, E2F1
rons of patients with mid-stage Alzheimer’s disease, who have and c-Myb synergistically transactivate PAX6. The clinical rele-
neurofibrillary tangles (GEO Profile Record GDS2795).46 Another re- vance of the amyloid-b-induced upregulation of this pathway is
port suggested that PAX6 expression gradually increases in the corroborated by microarray data obtained from post-mortem
hippocampi of patients with Alzheimer’s disease as the disease brains of Alzheimer’s disease patients and a transgenic APP mouse
progresses, with a 1.2-times increase in incipient, 1.3-times in- model. Furthermore, RNAi-mediated downregulation of either
crease in moderate and 1.8-times increase in severe Alzheimer’s PAX6 or c-Myb protects cortical neurons from amyloid-b-induced
disease cases.47 c-Myb expression follows the same pattern, with a cell death, indicating that both genes have proapoptotic roles.
3.1-times increase for incipient cases, 2.9-times increase for mod- Importantly, we identified a potential target gene for PAX6, GSK3B
erate cases and 5.1-times increase for severe cases (GEO Profile (GSK-3b), one of the main kinases that phosphorylates tau protein,
Record GDS810, GDS4136). These findings suggest that PAX6 and possibly leading to the formation of neurofibrillary tangles. We
c-Myb expression is upregulated throughout the course of showed that amyloid-b neurotoxicity causes PAX6 to transactivate
Alzheimer’s disease. GSK-3b, the upregulation of which was seen in multiple models of
We provide in vitro data supporting a plausible mechanism Alzheimer’s disease, as well as in post-mortem human
through which amyloid-b activates the cellular signalling path- Alzheimer’s disease brains.48,49 Downregulation of PAX6 blocked
ways involving CDK, pRB and E2F1 by activating downstream the amyloid-b-induced GSK-3b increase and tau phosphorylation.
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Figure 6 Proposed model for the role of PAX6 in amyloid-b induced neurotoxicity.

This interaction indicates a potential signalling pathway linking CDK5, protein kinase A, MAPK) and phosphatases (protein phos-
amyloid-b to tau pathology. However, it is known that inhibitory phatase 2A and 2B) contribute to tau hyperphosphorylation and
serine-phosphorylation also regulates GSK-3b activity. And in this neurofibrillary tangles formation,44,51,52 there is still a missing link
study, the effect of PAX6 on GSK-3b phosphorylation needs to be in understanding the mechanism of this regulation. GSK-3b, a ser-
further investigated. ine/threonine, proline-directed kinase is involved in a diverse
Evidence supports the idea that cell cycle re-entry in terminally array of signalling pathways, and strongly implicated in
differentiated neurons causes neuronal death.50 However, how cell Alzheimer’s disease pathogenesis. GSK-3b can phosphorylate tau
cycle signals actually trigger neuronal death is largely unknown. at multiple serine residues, and both its protein level and kinase
E2F1 has been shown to regulate amyloid-b-associated neuronal activity are increased in Alzheimer’s disease.42,53 Also, GSK-3b
death dependent on a classic mitochondrial pathway including inhibitors have been shown to reduce Alzheimer’s disease path-
Bcl-2-associated X protein and caspase-3.19 Considering the proa- ology in vivo and in preclinical trials.54,55 Importantly, our data
poptotic role of the transcription factors involved in the identified showed a novel pathway for hyperphosphorylation of tau protein.
pathway, raises the more specific question of how the E2F1/c-Myb/ Although we focused on the hyperphosphorylation of tau protein
PAX6 pathway triggers caspase activation. To address this ques- in this study, we also observed that total tau was reduced after
tion, additional downstream targets for these transcription factors Pax6 knockdown in RNA and protein level (Supplementary Table 2
need to be identified and characterized. and Fig. 5G). Whether PAX6 regulates total tau directly or indirectly
The biological activity of tau is modulated by its phosphoryl- needs further study.
ation status. Although previous studies have shown that post- When first identified as Alzheimer’s disease-associated factors,
translational modifications by multiple kinases (GSK-3b, CDK1 and the cause-effect relationship between amyloid-b and tau was not
PAX6 links between amyloid-b and p-tau BRAIN 2021: 144; 2759–2770 | 2769

clear, but extensive studies have been performed in that regard. 3. Bloom GS. Amyloid-beta and tau: The trigger and bullet in
For example, Amar et al.56 show that amyloid-b evoked an increase Alzheimer disease pathogenesis. JAMA Neurol. 2014;71(4):
in tau phosphorylation dependent on CaMKIIa activation. This 505–508.
interaction is supported by our RNA-sequencing data and seems to 4. Kant R, Goldstein LSB, Ossenkoppele R. Amyloid-b-independent
also involve Pax6 (Supplementary Table 1). Additionally, our RNA- regulators of tau pathology in Alzheimer disease. Nat Rev
sequencing data (Supplementary Table 2) indicated that Pax6 Neurosci. 2020;21(1):21–35.
silencing in neurons could significantly decrease the expression 5. He Z, Guo JL, McBride JD, et al. Amyloid-beta plaques enhance
levels of several key kinases that phosphorylate tau, such as Cdk5 Alzheimer’s brain tau-seeded pathologies by facilitating neurit-
(17.3%) and Mapk1 (63.8%). Therefore, there are probably more tar- ic plaque tau aggregation. Nat Med. 2018;24(1):29–38.
gets and downstream pathways of PAX6 involved in Alzheimer’s 6. Moh C, Kubiak JZ, Bajic VP, Zhu X, Smith MA, Lee HG. Cell cycle
disease pathogenesis. deregulation in the neurons of Alzheimer’s disease. Results
In summary, our study provides evidence that amyloid-b Probl Cell Differ. 2011;53:565–576.
neurotoxicity leads to hyperphosphorylation of tau through acti- 7. Bonda DJ, Lee HP, Kudo W, Zhu X, Smith MA, Lee HG.
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activation in neurons, subsequent activation of transcription fac-
8. Ding XL, Husseman J, Tomashevski A, Nochlin D, Jin LW,
tors such as c-Myb and PAX6 and hyperactivation of GSK-3b. The
Vincent I. The cell cycle Cdc25A tyrosine phosphatase is acti-
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Constitutive Cdc25B tyrosine phosphatase activity in adult
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brain neurons with M phase-type alterations in Alzheimer’s
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therapeutic strategy is to combine amyloid-b removal and target-
10. McShea A, Harris PL, Webster KR, Wahl AF, Smith MA.
ing PAX6 to prevent neuronal death and tau hyperphosphoryla-
Abnormal expression of the cell cycle regulators P16 and CDK4
tion, therefore to slow Alzheimer’s disease progression. The
in Alzheimer’s disease. Am J Pathol. 1997;150(6):1933–1939.
pathway that we identified suggests E2F1, c-Myb or PAX6 as novel
11. Giovanni A, Wirtz-Brugger F, Keramaris E, Slack R, Park DS.
targets for pharmaceutical intervention.
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We thank Changyuan Song for lifelong support for research in phosphorylated retinoblastoma protein is associated with tau
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70(7):578–587.
13. Vincent I, Jicha G, Rosado M, Dickson DW. Aberrant expression
Funding of mitotic cdc2/cyclin B1 kinase in degenerating neurons of
This work was supported by grants from the National Natural Alzheimer’s disease brain. J Neurosci. 1997;17(10):3588–3598.
Science Foundation of China (No. 81271226), the seed funding of 14. Smith MZ, Nagy Z, Esiri MM. Cell cycle-related protein expres-
the University of Hong Kong, and the Research Grant council of the sion in vascular dementia and Alzheimer’s disease. Neurosci
Hong Kong Special Administrative Region (No. 11200218). E.F.F. Lett. 1999;271(1):45–48.
was supported by HELSE SØR-ØST (#2017056, #2020001, #2021021), 15. Ueberham U, Hessel A, Arendt T. Cyclin C expression is
the Research Council of Norway (#262175 and #277813), the involved in the pathogenesis of Alzheimer’s disease. Neurobiol
National Natural Science Foundation of China (#81971327), and an Aging. 2003;24(3):427–435.
Akershus University Hospital Strategic grant (#269901). 16. Arendt T, Rodel L, Gartner U, Holzer M. Expression of the cyclin-
dependent kinase inhibitor p16 in Alzheimer’s disease.
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Competing interests 17. Arendt T, Holzer M, Gartner U. Neuronal expression of cycline
dependent kinase inhibitors of the INK4 family in Alzheimer’s
E.F.F. has CRADA arrangements with ChromaDex and is a consult-
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ant to Aladdin Healthcare Technologies and the Vancouver
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Dementia Prevention Centre.
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