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Mark4

ablation attenuates pathological phenotypes in a mouse model of tauopathy

Grigorii Sultanakhmetov

, Sophia Jobien M. Limlingan

, Aoi Fukuchi

, Keisuke Tsuda

Hirokazu Suzuki

, Iori Kato

, Taro Saito

1,2

, Adam Z. Weitemier

1,2

, and Kanae Ando

1,2,*

Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan

University, Tokyo 192-0397, Japan

Department of Biological Sciences, School of Science, Tokyo Metropolitan University, Tokyo

192-0397, Japan

Corresponding author:

Kanae Ando

1-1 Minamiosawa, Hachioji, Tokyo, Japan 192-0397

k_ando@tmu.ac.jp

Short title:

Mark4

ablation mitigates tauopathy

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Abstract

Accumulation of abnormally phosphorylated tau proteins is linked to various

neurodegenerative diseases, including Alzheimer’s disease and frontotemporal dementia.

Microtubule affinity-regulating kinase 4 (MARK4) has been genetically and pathologically

associated with Alzheimer’s disease and reported to enhance tau phosphorylation and toxicity

Drosophila

and mouse traumatic brain-injury models but not in mammalian tauopathy

models. To investigate the role of MARK4 in tau-mediated neuropathology, we crossed P301S

tauopathy model (PS19) and Mark4 knockout mice. We performed behavior, biochemical, and

histology analyses to evaluate changes in PS19 pathological phenotype with and without

Mark4. Here, we demonstrated that

Mark4

deletion ameliorated the tau pathology in a mouse

model of tauopathy. In particular, we found that PS19 with

Mark4

knockout showed improved

mortality and memory compared with those bearing an intact

Mark4

gene. These phenotypes

were accompanied by reduced neurodegeneration and astrogliosis in response to the reduction

of pathological forms of tau, such as those phosphorylated at Ser356, AT8-positive tau, and

thioflavin S-positive tau. Our data indicate that MARK4 critically contributes to tau-mediated

neuropathology, suggesting that MARK4 inhibition may serve as a therapeutic avenue for

tauopathies.

Keywords

Mark4

, Alzheimer’s disease, tauopathy, neurodegeneration, astrogliosis

Introduction

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The abnormally phosphorylated form of the microtubule-associated protein tau has been

identified as a major component of paired helical filaments (PHFs) in neurofibrillary tangles

(NFTs) and plaque neurites in the brains of patients with Alzheimer’s disease (AD).

1–3

NFT

depositions have been associated with cognitive decline and pathology severity in AD,

4,5

which

are the most prevalent causes of aging-associated dementia

. Under physiological conditions,

tau regulates microtubule stability in the axon, whereas in disease, it is hyperphosphorylated

and aggregates.

Among its many phosphorylation sites, those located in the tau microtubule-binding

repeats, such as Ser262 and Ser356, regulate its physiological and pathological functions.

High

Ser262 phosphorylation has been observed from early-stage AD disease in pre-NFT neurons

and correlates with the propagation of tau pathology.

Tau phosphorylation at Ser262 and

Ser356 was previously shown to promote phosphorylation at other AD-associated phospho-

epitopes such as AT100 (phosphorylation at Thr212 and Ser214) and AT8 (phosphorylation at

Ser202 and Thr205).

Tau phosphorylation at Ser262 and Ser356 affects its interactions with

chaperone complexes and degradation,

12,13

intracellular distribution,

and liquid-liquid phase

separation.

Substitution of these sites by non-phosphorylatable alanines dramatically reduces

tau toxicity in

Drosophila

models,

11,16,17

suggesting that phosphorylation at these sites is critical

for tau toxicity.

MARK4 belongs to the Par-1/microtubule affinity-regulating kinase (MARK) family,

which constitutes evolutionarily conserved Ser/Thr kinases that phosphorylate microtubule-

associated proteins, including tau, to regulate microtubule-dependent transport and stability.

18–

Previous studies reported that MARK3 and MARK4 are sequestered to granulovacuolar

degeneration bodies along with tau phosphorylated at Ser262 in patients with AD,

and

elevation of the MARK4-tau interaction correlates with Braak stages.

Moreover, genomic

studies showed that a

Mark4

de novo genetic variant was linked to early-onset AD,

and AD-

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linked single-nucleotide polymorphisms were identified within the

Mark4

gene by a Bayesian

genome-wide association study.

Drosophila

, Par-1 overexpression enhances human tau

toxicity, whereas Par-1 suppression mitigates it.

11,26

Previously, we showed that expression of

human

MARK4

also enhances tau toxicity in a

Drosophila

model,

27,28

suggesting mediation of

tau abnormality by MARK4. Other studies have uncovered additional

Mark4

functions in

mouse models of obesity

and ischemic brain injury.

Nevertheless, the role of MARK4 in a

mammalian tauopathy model has not been investigated.

Here, we examined MARK4 involvement in the disease pathogenesis of a tauopathy

mouse model (PS19). By combining PS19 with a

Mark4

knockout genetic background, we

demonstrated that

Mark4

deficiency significantly improved the lifespan and memory of the

PS19 model. We found that although

Mark4

knockout did not affect tau phosphorylation at

Ser262, it decreased Ser356 phosphorylation and reduced the abundance of AT8 phospho-

epitopes and thioflavin S-positive aggregates. Interestingly,

Mark4

deletion mitigated

astrogliosis in the brains of both PS19 and aged non-transgenic mice. Our results demonstrate

that lowering MARK4 levels is sufficient to ameliorate the tauopathy phenotype in a mouse

model, suggesting its critical involvement in neurodegenerative pathology.

Materials and methods

The study was approved by the Research Ethics Committee of Tokyo Metropolitan University

(approval numbers: A5-5, A5-6, A4-6, A4-23, and A3-11).

All animal experiments were

performed according to the Tokyo Metropolitan University animal experimentation guidelines

and Science Council of Japan guidelines.

Animals

Mark4

knockout-mouse cryo-preserved spermatozoa (strain name: C57BL/6NCrl-

Mark4

em1(IMPC)

Mbp/Mmucd, RRID: MMRRC_043405-UCD) were purchased from the Mutant

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Mouse Resource and Research Center at the University of California at Davis. In this strain,

exons 3, 4, and the flanking splicing regions of the

Mark4

gene have been deleted using

CRISPR/Cas9 gene editing. Litters were recovered by RIKEN BioResource Research Center

(Tsukuba, Ibaraki, Japan). Mice expressing the human 1N4R tau protein bearing the

frontotemporal dementia-associated P301S mutation (PS19; strain name: B6;C3-Tg (Prnp-

MAPT*P301S) PS19Vle/J, RRID: IMSR_JAX:100010) under the prion promoter were

purchased from Jackson Laboratories (Bar Harbor, Maine, the United States). We used

heterozygous PS19 male mice in this study.

All mice were bred in the Tokyo Metropolitan University animal facility in a special pathogen-

free area with a 12:12 h light/dark cycle and free access to food (Picolab mouse diet 20, 5058)

and water.

Mark4

knockout and PS19 mice were crossed to obtain littermates

Mark4

+/+

(wild

type [WT]),

Mark4

+/-

Mark4

-/-

, PS19, PS19:

Mark4

+/-

, and PS19:

Mark4

+/-

. The first generation

was obtained by crossing PS19 with

Mark4

+/-

and the second was obtained by crossing

PS19:

Mark4

+/-

with

Mark4

+/-

. We used first-generation littermates for the survival assay and

second-generation littermates for the behavioral, histological, and biochemical assays

(breeding scheme: Figure 1A). Genotypes were confirmed by PCR of tail DNA according to

the manufacturer’s genotyping protocols using the primers listed in Table 1. Ataxia, or reaching

the age of 12 months, was considered an endpoint in survival experiments. Mice that developed

ataxia were not used in any experiments. For histological experiments, mice were deeply

anesthetized with a double dose of 0.3 mg/kg medetomidine, 4.0 mg/kg midazolam, and 5.0

mg/kg butorphanol tartare.

Behavioral tests

PS19 mice are known to manifest cognitive impairments at the age of 6 months.

Mice of

different genotypes were randomly assessed on different experimental days. Experiments were

performed during the light cycle in a space with dim illumination by experimenters blinded to

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mice genotypes on the experimental day and during analysis for all the behavioral assays. For

behavioral assays, mice were habituated to the experimental space at least 30 min before assay

initiation.

Open field assay

Mice were allowed to explore a novel white acrylic 40×40×40 cm box for 10 min. The activity

was recorded on a 720p web camera. Tracking software (ToxTrack) was used to evaluate the

travel distance and time spent in a 20x20 cm middle area of the arena. The box was wiped with

70% EtOH prior to usage by each individual mouse. We excluded mice from experiments

(approximately 1–3 per genotype) that showed abnormal behavior such as intensive jumping,

tail rattling, and/or sitting at a corner for more than half of the experimental time. The open

field test was considered a habituation phase for the novel object recognition assay, which was

performed the next day.

Novel object recognition assay

Experiments were performed according to a previously published protocol.

Briefly, the day

after the open field assay, mice were placed in the arena containing two identical objects. They

were allowed to explore either or both objects for a total of 20 seconds within a maximum 10-

minute session. Twenty-four hours later, mice were placed in the arena containing one familiar

and one novel object and again allowed to explore for 20 seconds within a 10-minute session.

Mice were considered to explore objects when their nose was headed to the object’s direction

at a 2-cm distance. Then, the time spent exploring each object was analyzed. The discrimination

index (DI) was calculated using the following equation:

𝐷𝐼

𝑇

𝑛𝑜𝑣𝑒𝑙

― 𝑇

𝑜𝑙𝑑

𝑇

𝑛𝑜𝑣𝑒𝑙

𝑇

𝑜𝑙𝑑

where T

novel

and T

old

are the times spent observing novel and old objects, respectively. Mice

that did not show any interest in objects, i.e., they explored all objects for less than 20 seconds

within a 10-minute session, were excluded from the experimental analysis.

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Y-maze assay

We observed mouse behavior in the Y-maze to evaluate spatial working memory in our

transgenic mice, as previously described.

Briefly, we placed a mouse into the center of the

Y-maze and allowed it to observe the maze for 10 min. The video was recorded for every mouse

to analyze their behavior. The Y-maze has 35 cm arms with 10 cm height and 5 cm width. A

successful alternation between arms is recorded when mice do not return to previously explored

arms, and the percentage of successful alternation (%SA) was calculated using the following

equation:

%𝑆𝐴

𝐴

𝑠𝑢𝑐𝑐𝑒𝑠𝑠𝑖𝑣𝑒

𝐴

𝑡𝑜𝑡𝑎𝑙

―

where A

successive

and A

total

represent the amounts of successive and total alternations,

respectively. We subtracted the first two alternations because they could not be successful or

wrong. Mice that climbed onto walls or jumped out from the maze were excluded from the

analysis.

Histology

Mice were deeply anesthetized with a double dose of 0.3 mg/kg medetomidine, 4.0 mg/kg

midazolam, and 5.0 mg/kg butorphanol tartare.

Then, mice were perfused with ice-cold

phosphate-buffered saline (PBS) following 4% paraformaldehyde (PFA)/PBS. Mouse brains

were extracted and fixed in 4% PFA/PBS solution for 24 h. Next, brains were immersed in

30% sucrose in PBS until the brain sunk to the bottom of the 15-mL tube. Brains were sliced

40-μm

sections using a Leica cryostat CM 1510 S (Wetzlar, Germany). The sections were

immersed in cryoprotectant solution (30% ethylene glycol and 20% glycerol in PBS) and kept

at -20°C until further use. Mouse brain slices were observed using a Keyence BZ-X710 (Osaka,

Japan) epifluorescence microscope and a Nikon AX/AX R confocal microscope (Tokyo,

Japan).

Immunofluorescence

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We performed standard free-floating mouse brain section staining to examine the effect of

Mark4

ablation on P301S tau levels, gliosis, and neurodegeneration in PS19 mice.

Briefly,

mouse brain sections were washed in PBS, followed by permeabilization with 0.1% Triton X-

100. Samples were blocked in 5% normal goat or donkey serum, depending on the host in

which the secondary antibodies used were raised, for 1 h at room temperature. Then, sections

were incubated with primary antibodies overnight at 4°C, followed by incubation for 2 h with

secondary antibodies at room temperature and counterstaining with DAPI. Samples were stored

in the dark at 4°C prior to being imaged.. The primary and secondary antibodies used are listed

in Table 2.

Thioflavin S staining

Thioflavin S staining was performed to analyze tau tangles in the brains of tauopathy model

mice upon MARK4 protein ablation. Briefly, after washing, slices were mounted on a glass

slide and allowed to completely dry on a heat plate at 42°C, followed by incubation with 0.5

mM thioflavin S (Merck, T1892) in 50% ethanol for 7 min.

35,36

The number of thioflavin S-

positive puncta was quantified using default thresholding segmentation and measure-particle

functions in ImageJ (NIH).

Biochemical analysis

At nine months of age, mice were sacrificed by cervical dislocation, and their brains were

collected, snap-frozen in liquid nitrogen, and kept until further use at -80°C.

In vitro kinase assay

In vitro

kinase assay

was performed

to validate kinase activity levels in our

Mark4

knockout

mice. Mouse whole brains were homogenized in 10 vol (1:10 weight/volume ratio) of 3-

morpholino-propane-sulfonic acid (MOPS) buffer (20 mM MOPS, pH 6.8, 1 mM EGTA, 0.1

mM EDTA, 0.3 M NaCl, 1 mM MgCl

, 0.5% Nonidet P-40, and 1 mM DTT + proteinase

inhibitors: 0.2 mM Pefabloc SC, and 1 mg/mL leupeptin, and phosphatase inhibitors: 10 mM

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

-glycerophosphate and 5 mM NaF) with a Teflon pestle homogenizer and centrifuged at

10,000×g for 15 min at 4°C to collect the extract as a supernatant. MARK4 was

immunoprecipitated from brain lysates with an anti-MARK4 antibody (Novus Biologicals,

NB100-1013) and protein-G Dynabeads (Thermo Fisher Scientific). Its kinase activity was

measured using Chktide (SignalChem) and [g-

P]ATP as substrates. The incorporation of

into Chktide was quantified using a liquid scintillation counter (Beckman Coulter).

Western blotting

Western blots were performed to analyze the relative protein levels of total and fractionated

tau. One of the two brain hemispheres was homogenized in 10 vol of radioimmunoprecipitation

assay (RIPA) buffer (50 mM Tris base, pH 8.0, 0.15 M NaCl, 1% Nonidet P-40, 0.5% Na-

deoxycholate, 5 mM EDTA, 1 mM EGTA, 0.1% SDS + proteinase inhibitors and phosphatase

inhibitors) using a Teflon pestle homogenizer and centrifuged at 15,800×g for 20 min at 4°C

to collect the extract as a supernatant. Protein concentration was measured by Bradford protein

assay (Pierce, 23200)

and adjusted to 2

µg/μL.

Then, protein homogenates were mixed with

2× loading buffer (124.8 mM Tris base pH 6.8, 4% SDS, 20% glycerol, 0.2 mg/mL

bromophenol blue, 10% 2-mercaptoethanol + proteinase inhibitors and phosphatase inhibitors)

to a final concentration of 1

µg/μL,

and 10

μg

of protein from each sample was loaded to a

10% gel for

SDS‒PAGE.

After gel electrophoresis, proteins were transferred to PVDF

membranes, which were blocked in 5% bovine serum albumin/TBST (0.1 M Tris base pH 7.6,

0.15 M NaCl, 0.05% Tween 20) and then incubated with the designated primary and secondary

antibodies described in Table 2. The band signals were visualized using Immobilon Western

Chemiluminescent HRP Substrate (Millipore) in Fusion SL (Vilber, France).

Tau protein extraction

Sequential extraction of tau protein was performed to test the effect of MARK4 on tau protein

solubility. We performed fractionation analysis as previously described for PS19 mice.

34,37

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Briefly, one brain hemisphere was homogenized in 5 vol high-salt reassemble buffer (HS-RAB:

100 mM MES, pH 7.0, 1 mM EGTA, 0.5 mM MgSO

, 0.75 M NaCl, 0.1 mM EDTA +

proteinase inhibitors and phosphatase inhibitors ) using a Teflon pestle homogenizer, and the

homogenate was centrifuged at 50,000×g in an Optima MAX-TL ultracentrifuge (Beckman

Coulter, Brea, CA, the United States) for 40 min at 4°C to collect the supernatant as an HS-

RAB soluble fraction. The pellet was homogenized in 1 M sucrose/RAB buffer and the solution

was centrifuged at 50,000×g for 20 min, followed by pellet homogenization in 1 vol RIPA

buffer and centrifugation at 50,000×g for 20 min at 4°C to collect the supernatant as a RIPA

soluble fraction. Next, we extracted a RIPA-insoluble pellet with 1 vol of cold 70% formic acid

solution and centrifuged it at 15,800×g for 20 min at 4°C to collect the supernatant as an FA

soluble fraction. The FA fraction was diluted in 1:10 (v/v) neutralization buffer (1 M Tris base,

0.5 M Na

HPO

), and the pH was checked using pH strips (Merck). All fractions were

processed for western blotting as described above.

Analysis

Experimenters were blind regarding mice genotype during data collection and manual analysis.

The study design was made to create a random distribution regarding genotype for data and

sample collection. Briefly, we crossed PS19:

Mark4

+/-

and

Mark4

+/-

to get littermates with

different genotypes, which we split 2-4 per cage in random order; therefore, when mice reached

the desired age for experiments, they were assayed or collected in random order regarding

genotype. For the open field test, the traveled distance and time spent in the middle area (20×20

cm) were quantified using ToxTrack version 2.96.

38,39

Western blot and microscopy data were

analyzed using ImageJ version 1.53c.

40,41

Band intensities were quantified using gel-selection

and plot-line functions followed by band peak underline area measurements. In

immunohistochemistry experiments, the area covered by astroglia and microglia was quantified

using default thresholding segmentation and measured-particle functions. For other signals,

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integrated intensities were evaluated using the measurement function, followed by

normalization to the region of interest size. Data are represented as fold changes, normalized

to average levels of control (PS19 or WT groups).

Statistical analysis

Data analysis was performed in GraphPad Prism 9 and GPower 3.1. We computed the required

total sample size of 54 for behavioral experiments (medium effect size

desired

𝑓

= 0.5

power

), which means nine mice per experimental group on average.

― 𝛽

= 0.9

𝛼

= 0.05

Our “dummy” test for histological analysis predicted five mice per genotype for 0.9 power –

the actual power was between 0.8 and 0.9 for experiments that showed differences among

means. The Mantel-Cox test was used to compare survival curves between PS19 and

PS19:

Mark4

+/-

animals. A two-way analysis of variance (ANOVA) test was used for the

behavioral assay, where

Mark4

deficiency was analyzed in WT and PS19 mice followed by a

Tukey’s multiple comparisons test of the means between each group. For other tests, in which

the effect of

Mark4

deficiency was analyzed in PS19 mice, and WT was used as a negative

control, one-way ANOVA was implemented, followed by Holm-Sidak’s or Dunnett’s multiple

comparison tests among the means or between the means of the PS19 and the PS19:

Mark4

+/-

or PS19:

Mark4

-/-

groups, respectively. Data normality and homogeneity were tested by

D'Agostino-Pearson omnibus (K2) and Barlett’s (one-way ANOVA) or Spearman’s (two-way

ANOVA) tests, correspondingly. For thioflavin S staining analysis, we performed a

Kruskal‒Wallis

test followed by a Dunn’s multiple comparisons test. Statistical tests for each

experiment are specified in the figure legends. Differences were considered statistically

significant when

𝑝

< 0.05

Results

Mark4 knockout prolonged lifespan and rescued memory deficits in PS19 mice

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To test whether MARK4 suppression affects the abnormal behavioral phenotype and

mortality of PS19 mice, we crossed them with

Mark4

knockout mice (Figure 1A, two

generations F1 and F2). Homozygous

Mark4

knockout mice (hereafter referred to as

Mark4

-/-

)

are fertile and develop to adulthood without apparent abnormalities.

43,44

In addition to tail PCR

genotyping, we confirmed the absence of MARK4 in

Mark4

-/-

mice by western blotting and

vitro

kinase assays (Figure 1B, C, S1). PS19 mice have a shorter lifespan,

and in our colony,

they had a median lifespan of 10.9 months, while all WT and

Mark4

+/-

mice survived until 12

months of age. We found that in the first generation from crossing PS19 and

Mark4

+/-

mice

(F1, Figure 1A) PS19:

Mark4

+/-

mice lived significantly longer than their PS19 counterparts

(

, Figure 1D): at 12 months, the survival rates of PS19 and PS19:

Mark4

+/-

were 16%

𝑝

< 0.001

vs. 65%, respectively. PS19:

Mark4

+/-

and PS19:

Mark4

-/-

obtained by crossing PS19:

Mark4

+/-

and

Mark4

+/-

(F2, Figure 1A) showed less number of mice developing ataxia compared to

PS19 by 9 months old (Table S1).

Hyperactivity of PS19 mice, such as enhanced locomotion and more frequent

alternations in the Y-maze,

as well as enhanced locomotor activity in the open field test,

32,45

has been previously reported. In agreement with previous studies, 8-month-old PS19 mice

showed enhanced locomotor activity compared with WT mice in the open field test.

PS19:

Mark4

-/-

mice showed a shorter, albeit not significantly, traveled distance than PS19 mice

(

Figure 1E) and spent a similar amount of time in the middle area of the arena as

𝑝

= 0.46,

PS19 mice (Supplementary Figure 2A, B).

To assess the memory performance of our transgenic mice, we implemented the Y-

maze spontaneous alteration (SAT) and novel object recognition (NOR) tests. The Y-maze

SAT is designed to evaluate spatial working memory in mice.

As previously reported,

32,45

PS19 mice showed a lower number of correct alternations. Interestingly,

Mark4

copy number

negatively correlated with increased performance of PS19 mice in the Y-maze SAT (57%, 63%,

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71% of correct alternations in PS19, PS19:

Mark4

+/-

, and PS19:

Mark4

-/-

mice, respectively),

with a significant difference between PS19 and PS19:

Mark4

-/-

mice (

Figure 2F).

𝑝

< 0.05,

However, the number of total alternations in the Y-maze was not significantly different

between experimental groups (Figure S2C). To assay recognition memory in our transgenic

mice,

we performed the NOR test and observed that PS19 mice performed more poorly than

WT in agreement with previous studies (

32,45

and Figure 1G). PS19:

Mark4

+/-

or PS19:

Mark4

-/-

performed better than PS19: the NOR memory performance scores of PS19:

Mark4

+/-

and

PS19:

Mark4

-/-

were higher than those of PS19 (

and

, respectively) and

𝑝

= 0.063

𝑝

= 0.044

similar to those of WT (

and

, respectively, Figure 1G).

𝑝

= 0.99

𝑝

= 0.99

In addition, we did not observe abnormalities in the survival, open field activity, or

memory functions of our

Mark4

knockout mice (Figure 1D–G; WT,

Mark4

+/-

, and

Mark4

-/-

groups). Our findings corroborate previous studies, which did not identify pathological

abnormalities in

Mark4

deficient mice.

29,43,44

Mark4 knockout ameliorated the loss of synapses and dendrites in PS19 mice

We were then interested in whether

Mark4

knockout affects neurodegeneration in PS19

mice. To this end, we analyzed neurons and glia in regions involved in memory functions in

the hippocampus, amygdala, and piriform cortex, where we confirmed a relatively high level

of human P301S tau protein in the brain of a nine-month-old PS19 mouse (Supplementary

Figure 3) as was also shown in previous studies.

34,36,47

First, we performed immunostaining

using an antibody against the postsynaptic marker PSD-95 to identify synapse density. We

observed that synapse density was lower in all tested brain regions of PS19 mice (

𝑝

< 0.05,

Figure 2A, B, C, and Supplementary Figure 4; compare WT and PS19). Although

PS19:

Mark4

+/-

brains exhibited only moderate changes in PSD-95 immunoreactivity compared

with PS19 animals (

, Figure 2A, B, C), PS19:

Mark4

-/-

brains displayed significantly

𝑝

> 0.1

higher PSD-95 signal in the amygdala and piriform cortex than PS19 brains (

), and

𝑝

< 0.05

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the signals were as high as those in WT (

and

, respectively, Figure 2A, B,

𝑝

= 0.97

𝑝

= 0.85

C). The average PSD-95 staining intensity in PS19:

Mark4

-/-

mice was also higher in the

hippocampus than in PS19 mice (

𝑝

= 0.065

Immunostaining with MAP2 antibody was carried out to detect dendritic loss.

Among

the tested regions, MAP2 immunoreactivity was similar in the hippocampus and amygdala

between all experimental groups (

, Figure 2A, C). MAP2 significantly reduced only

𝑝

> 0.5

in the piriform cortex of PS19 mice compared with the WT (

), whereas it was

𝑝

< 0.01

recovered in PS19:

Mark4

-/-

animals compared to PS19 (

, Figure 2A, B, C, and

𝑝

= 0.056

Supplementary Figure 4).

Mark4 knockout reduced Ser356 phosphorylation levels in PS19 mice

MARK4 phosphorylates the

KXGS

motifs of the tau microtubule-binding repeats, such

as at Ser262 and Ser356.

Therefore, we sought to identify whether

Mark4

ablation affects tau

phosphorylation at Ser262 and Ser356 (pSer262 and pSer356) in PS19 mice. Immunostaining

with a tau anti-pSer262 antibody showed no differences among PS19, PS19:

Mark4

+/-

, and

PS19:

Mark4

-/-

genotypes in all tested regions (

, Figure 3A, B). pSer356

𝑝

> 0.1

immunolabeling was weak in CA1 and prevalent in CA3 and the dentate gyrus in the

hippocampus of PS19 mice (Figure 3C, insets). By contrast to pSer262, pSer356 signal

intensity was reduced about two-fold in PS19:

Mark4

+/-

and PS19:

Mark4

-/-

compared with PS19

in all tested regions (Figure 3C, D). The reduction in pSer356 was significant for hippocampus

and piriform cortex regions in PS19:

Mark4

-/-

and PS19:

Mark4

+/-

compared to PS19 (

𝑝

< 0.05

Figure 3D).

Additionally, we analyzed human tau expression with the human tau-specific antibody

HT7. Immunostaining with HT7 exhibited significantly higher signal intensity in the

hippocampus of PS19:

Mark4

-/-

mice than in PS19 animals (

), whereas it displayed

𝑝

< 0.05

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similar intensity in the amygdala and piriform cortex (

, Figure 3E, F). PS19:

Mark4

+/-

𝑝

> 0.1

mice showed similar levels of HT7 tau as PS19 mice (

, Figure 3E, F).

𝑝

> 0.1

Western blotting experiments confirmed the reduction of pSer356 in PS19:

Mark4

-/-

compared to PS19 (

, while pSer262 levels were not significantly affected (

𝑝

< 0.05)

𝑝

> 0.1

Figure 3G, H, S5, pSer356, pSer262). Total tau levels were not significantly affected by

MARK4 knockout in PS19 mice (

, Figure 3G, H, Supplementary Figure 5, HT7).

𝑝

> 0.1

Mark4 knockout reduced AT8-positive tau levels in PS19 mice

Immunostaining intensity using an AT8 antibody (pSer202, pThr205) correlates with

disease severity in tauopathy.

PS19 mice start to exhibit an AT8 signal above baseline around

five months of age in the hippocampus.

We conducted immunohistochemistry and western

blotting to evaluate the effect of

Mark4

knockout on the abundance of the AT8 phospho-epitope

in PS19 mice. We observed lower AT8 staining intensity in PS19:

Mark4

-/-

than in PS19 mice

when heterozygous Mark4 knockout has no significant effect on AT8 levels (Figure 4A-C).

The reduction in AT8 was significant for all tested regions in PS19:

Mark4

-/-

(

). Next,

𝑝

< 0.05

we compared the AT8-positive total tau levels in brain lysates. In line with

immunohistochemistry experiments, a two-fold reduction in AT8 signal in PS19:

Mark4

-/-

mice

was observed by western blotting (

, Figure 4C, Supplementary Figure 6). AT8-

𝑝

< 0.01

positive tau levels appeared to be, on average, reduced in PS19:

Mark4

+/-

compared with PS19

mice; however, this difference was not significant (

, Figure 4C). Additionally, we

𝑝

> 0.1

confirmed the presence of

Mark4

knockout in our tested groups (Figure 4C).

Mark4 knockout reduced thioflavin S-positive tau aggregates in PS19 mice

Thioflavin S-positive tau depositions have been observed in the neocortex,

hippocampus, and amygdala at six months of age in PS19 mice.

Thus, we sought to evaluate

the effect of MARK4 suppression on NFT pathology in PS19 mice by performing thioflavin S

staining. Thioflavin S staining of tau appeared as puncta (Figure 5A, white arrows). Thioflavin

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S puncta were significantly fewer in PS19:

Mark4

-/-

than in PS19 mice in all brain regions tested

(

, Figure 5B). A reduction in Thioflavin S-positive puncta was observed in

𝑝

< 0.05

PS19:

Mark4

+/-

animals, but the difference was not significant due to variations among

individuals (

). In particular, PS19:

Mark4

+/-

mice showed almost two-fold reduction

𝑝

> 0.1

compared to PS19, and PS19:

Mark4

-/-

showed further reduction: whereas PS19 had more than

a hundred puncta, PS19:

Mark4

-/-

animals displayed only a dozen puncta in average (Figure

5B).

Next, we analyzed the effect of

Mark4

knockout on tau solubility by conducting

sequential protein extraction from brain lysates using buffers with different extraction strengths

(HS-RAB→RIPA→formic

acid, Figure 5C), followed by western blotting with the anti-human

tau antibody HT7.

34,37

Tau in brain homogenates of PS19:

Mark4

+/-

and PS19:

Mark4

-/-

mice

tended to be fractionated more in HS-RAB and less in RIPA or formic acid fractions compared

with PS19. Tau proteins in the HS-RAB fraction from PS19:

Mark4

+/-

and PS19:

Mark4

-/-

mice

were 40% and 28% more, respectively, whereas those in the RIPA fraction were 52% and 37%

less, than in PS19 samples, respectively (Figure 5C, D, Supplementary Figure 7). Tau proteins

from PS19:

Mark4

-/-

samples fractionated to the formic acid fraction and were 33% less than

those in PS19 brain lysates (Figure 5C, D). However, the difference was insignificant due to

the large variation among individuals (

𝑝

> 0.1

Mark4 knockout reduced astrogliosis in WT and PS19 mice

We also sought to analyze the effect of

Mark4

ablation on the activation of microglia

and astroglia, which are signs of neuroinflammation. PS19 mice show neuroinflammation

detected by gliosis at six months of age.

Immunostaining with the microglial marker Iba1

revealed that although the number of microglia was increased in PS19 compared with WT mice,

it was not significantly reduced upon

Mark4

ablation (

among PS19, PS19:

Mark4

+/-

𝑝

> 0.1

and PS19:

Mark4

-/-

mice, Figure 6A, B). Glial fibrillary acidic protein (GFAP), a marker of

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reactive astroglia, was increased in the PS19 amygdala and piriform cortex, indicating

astrogliosis. In contrast to microgliosis,

Mark4

knockout displayed a prominent effect on

astrogliosis, as GFAP signals were lower in PS19:

Mark4

+/-

and PS19:

Mark4

-/-

than those in

PS19 mice (Figure 6C, D). We observed astrogliosis reduction in both the PS19:

Mark4

+/-

and

PS19:

Mark4

-/-

amygdala compared with PS19 mice (

). The piriform cortex in

𝑝

< 0.05

PS19:

Mark4

-/-

also showed a reduction in astrogliosis (

). In the hippocampus, GFAP

𝑝

= 0.053

staining was similar among WT, PS19, PS19:

Mark4

+/-

, and PS19:

Mark4

-/-

mice (

𝑝

> 0.1

Figure 6B, D). We also analyzed the levels of GFAP in brain extracts by western blotting.

GFAP levels were significantly reduced in PS19:

Mark4

-/-

compared with PS19 mice

(

, Figure 6E, Supplementary Figure 8).

𝑝

< 0.001

Since MARK4 is directly involved in inflammation processes,

we were motivated to

test whether

Mark4

ablation suppresses astrogliosis without human tau expression. We

compared GFAP staining of reactive astroglia in the hippocampus of 9-month-old WT,

Mark4

+/-

, and

Mark4

-/-

mice (Figure 7A), where GFAP-positive astroglia was more abundant

than in other tested regions of WT mice (Figure 6C, D).

Mark4

+/-

mice showed the same

reactive astroglia levels as their WT counterparts (

, Figure 7B). The GFAP signal was

𝑝

> 0.5

reduced to less than 50% in

Mark4

-/-

mice than in WT mice (

, Figure 7B), indicating

𝑝

< 0.01

that MARK4 functions independently of tau toxicity in the activation of astroglia.

Discussion

In this study, we analyzed the role of MARK4 in tau-induced neurodegeneration by

crossing a tauopathy model (PS19)

32,34,45,51

with

Mark4

knockout mice. We showed that

Mark4

deletion could decrease mortality, ameliorate memory deficits, and reduce synapse and

dendritic loss in PS19 mice (Figures 1 and 2).

Mark4

deficiency decreased the levels of tau

phosphorylation at Ser356 and AT8 and Thioflavin-S positive aggregates (Figures 3–5). Tau

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solubility was also analyzed biochemically with sequential protein extraction (Figure 5C).

Although

Mark4

deficiency tends to reduce tau insolubility, the difference was not statistically

significant. The number of animals was limited, and analyses with more animals in the future

may help to conclude the effect of

Mark4

knockout on tau solubility. We also demonstrated

that

Mark4

knockout suppressed astrogliosis in the PS19 model (Figure 6) and in the

hippocampus of mice that did not express human tau protein (Figure 7), thus revealing a novel

role of MARK4 in astrogliosis. In all experiments, MARK4

-/-

background improved PS19

phenotypes more prominently than MARK

+/-

background, indicating dosage-dependent effects

(Figure 1-7).

Previous reports suggest that a reduction in tau phosphorylation correlates with a lower

accumulation of pathological aggregates.

36,45,51–53

As a Par-1/MARK protein family member,

MARK4 phosphorylates tau at the serine residues of KXGS motifs of microtubule-binding

repeats, including Ser262 and Ser356.

Tau phosphorylation at Ser262 was detected in pre-

NFT neurons when prominent staining was observed with the 12E8 antibody, which recognizes

both pSer262 and pSer356 tau.

This finding suggests that tau phosphorylation at these sites

promotes tau aggregation. However, tau phosphorylation at Ser262 has been reported to

prevent tau aggregation,

and phosphorylation sites that drive tau aggregation do not include

pSer262.

Interestingly, the effect of Ser356 phosphorylation on tau aggregation has only been

investigated in the presence of pSer262. Here, we found that

Mark4

knockout decreased the

pSer356 without affecting pSer262 levels in PS19 animals (Figure 3). pSer356 reduction was

correlated with less AT8-positive tau and fewer thioflavin S-positive tau aggregates (Figures 4

and 5). These results suggest that reduced tau phosphorylation at Ser356 may block its

aggregation. They are also in agreement with a previous study which demonstrated that the

NUAK Family Kinase 1 (NUAK1) exclusively phosphorylated Ser356, and its ablation

reduced NFT formation and rescued memory deficits and synaptic plasticity in PS19 mice.

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Interestingly, both MARK4 and NUAK1 ubiquitination appears to be controlled by the

ubiquitin-specific protease-9,

and they are both associated with AD.

22,23,51

We found that

Mark4

ablation caused a dramatic reduction in AT8-positive and

thioflavin S-positive tau (Figures 4 and 5). Although the AT8 sites, Ser202 and Thr205, are not

direct MARK4 phosphorylation targets,

MARK4 may affect their phosphorylation via other

kinases.

GSK3β

can phosphorylate more than 15 tau phosphorylation sites, including AT8 sites,

8,58–61

and is believed to be essential for tau toxicity

in vivo

AT8 sites are also targets of

Cdk5,

8,63

and MARK4 enhances tau phosphorylation mediated by Cdk5.

Drosophila

, it

was shown that tau phosphorylation at Ser262 and Ser356 by Par-1, the fly homolog of MARK,

primed tau hyperphosphorylation at

GSK3β

sites.

Primed tau phosphorylation was further

confirmed in mammalian primary neurons.

We observed a two-fold AT8 signal reduction in

PS19 mice upon

Mark4

ablation (Figure 4C), which may, in part, be due to the effects of a

lower level of primed phosphorylation caused by decreased tau phosphorylation at Ser356. Tau

phosphorylation at Ser356 affects its interactions with molecular chaperones that enhance tau

aggregation,

suggesting that altered interactions with molecular chaperones may contribute

to decreasing the insoluble levels of tau in a

Mark4

knockout background. Our results highlight

a critical role of tau phosphorylation at Ser356 upstream of tau aggregation

in vivo

Finally, we found that

Mark4

knockout reduced astrogliosis to wild-type levels in the

PS19 mouse model (Figure 6), which may also contribute to ameliorating tau toxicity. Previous

reports indicate that a reduction in microgliosis and astrogliosis attenuates brain atrophy

without changing tau pathogenic phosphorylation,

suggesting that gliosis affects the disease

phenotype downstream of tau pathological perturbations. Interestingly, microgliosis was not

significantly affected by

Mark4

knockout in PS19 mice (Figure 6), and

Mark4

deletion reduced

the number of active astroglia in the hippocampus of 9-month-old mice that did not express

human P301S tau (Figure 7). These findings point towards a physiological role of MARK4 in

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astroglia activation, independent of tau lesions. MARK4 has been reported to mediate the

activation of NLPR3 inflammasomes, which constitute critical signaling platforms in bone

marrow-derived macrophages of the innate immune system.

It has been reported that loss of

Mark2

facilitates activation of microglia in culture and in the mouse brain.

However, the

functions in MARK2 in the astrocyte have not been reported. While all MARK family members

are expressed in astrocytes (Human Protein Atlas proteinatlas.org,

), we observed that

MARK4 ablation decreased astroglia activation (Figures 6 and 7), suggesting functions of

MARK4 in astrocytes are not redundant to other members.

Mark4

ablation may ameliorate tau-

induced neurodegeneration by not only reducing pathological tau modifications but also by

suppressing astrogliosis. Further investigation of glial changes in response to MARK4

suppression in other neurodegenerative disease models may reveal novel aspects of the

mechanisms underlying astroglia activation in disease pathogenesis.

In this study, we demonstrated the critical role of MARK4 in tau-induced

neuropathology and indicated that reducing MARK4 activity is sufficient to ameliorate tau

pathology. In particular,

Mark4

knockout in PS19 mice prolonged survival and restored

memory to wild-type levels, which was accompanied by reduced synapses and dendritic loss,

disease-associated tau phosphorylation, tau aggregation, and astrogliosis. Our results suggest

that MARK4 is a reasonable target for the identification of novel tauopathy treatments.

Data availability

The datasets used and/or analyzed in this study are available from the corresponding author

upon request.

Competing interests

The authors declare that they have no competing interests.

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Funding

This work was supported by research grants from the Japan Science and Technology Agency,

grant number JPMJFS2139 (to GS),

the Takeda Science Foundation (to KA), a research award

from the Japan Foundation for Aging and Health (to KA), the Novartis Foundation (Japan) for

the promotion of Science (to KA), a Grant-in-Aid for Scientific Research on Challenging

Research (Exploratory) [JSPS KAKENHI Grant number 19K21593] (to KA), and TMU

strategic research fund (to KA).

Acknowledgements

We thank Drs. Naruhiko Sahara (National Institutes for Quantum Science and Technology,

Chiba, Japan) for valuable advice on the tauopathy mouse model, Akiko Asada (Department

of Biological Sciences, Tokyo Metropolitan University) for technical help and comments, and

Shin-Ichi Hisanaga (Department of Biological Sciences, Tokyo Metropolitan University) for

critical comments on the manuscript.

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Page

tissues.

Sci Adv

. 2021;7(31):1-9. doi:10.1126/sciadv.abh2169

Figure Legends

Figure 1. MARK4 knockout prolonged lifespan and rescued memory deficits in PS19

mice.

(A)

Transgenic mouse breeding scheme and experimental timeline.

(B)

Western blot of

MARK4 from total brain lysates of WT (

Mark4

+/+

Mark4

+/-

, and

Mark4

-/-

mice. See

Supplementary Figure 1 for uncropped blots.

(C)

The

kinase activity of MARK4

immunoprecipitated from WT,

Mark4

+/-

, and

Mark4

-/-

mouse brain. The activity was measured

as counts per minute of [g-

P]ATP incorporation into substrate peptides. Data represent the

mean

standard deviation (SD).

(D)

Kaplan‒Meier

survival curve of PS19 compared with

PS19:

Mark4

+/-

from the first generation (F1) of cross PS19 and

Mark4

+/-

. The endpoint for

survival experiments was 12 months of age: 16% of the PS19 and 65% of PS19:

Mark4

+/-

mice

survived by this time. PS19 and PS19:

Mark4

+/-

curves were compared using a Mantel-Cox test

(***

(WT),

(

Mark4

+/-

(PS19), and

𝑝

= 0.0008

𝑁

= 15

𝑁

= 15

𝑁

= 19

𝑁

= 20

(PS19:

Mark4

+/-

(E)

Total distance traveled within 10 minutes in the open field test. Data

represent the mean

SD.

mice/group. Two-way Analysis of Variance

𝑁

= 8

𝑁

= 13

(ANOVA) was used to investigate the effect of the PS19 genotype (

). *

;

𝑝

= 0.0041

𝑝

< 0.05

Tukey’smultiple comparisons test.

(F)

Percentage of correct alteration in the Y-maze

spontaneous alteration test. Data represent the mean

SD.

mice/group.

𝑁

= 8

𝑁

= 11

Two-way ANOVA revealed the interaction effect between PS19 and

Mark4

knockout mice

(

). *

; Tukey’s multiple comparisons test.

(G)

The discrimination index

𝑝

= 0.0413

𝑝

< 0.05

was quantified to assess recognition memory in WT and PS19 mice in the presence or absence

Mark4

. Data represent the mean

SD.

mice/group. *

; two-way

𝑁

= 5

𝑁

= 10

𝑝

< 0.05

ANOVA with Tukey’s multiple comparisons test.

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Figure 2.

Mark4

knockout ameliorated the loss of synapses and dendrites in PS19 mice.

(A)

Representative images of double immunostaining of the hippocampus, amygdala, and

piriform cortex regions for the postsynaptic marker PSD-95 and dendritic marker MAP2. The

dashed line highlights the hippocampus (H), piriform cortex (PC), and amygdala (A). Insets

represent magnified CA1, CA3, amygdala (A), and piriform cortex (PC) regions. Scale bars,

500

μm

and 50

μm

(insets).

(B)

Representative laser scanning confocal microscopy images of

neurons in the piriform cortex for PSD-95 (magenta), MAP2 (green), and DAPI (blue).

Separate channel images are in Figure 3S. Scale bar, 10

μm.

(C)

Quantification of PSD-95 and

MAP2 integrated intensity in the hippocampus, amygdala, and piriform cortex normalized to

the region area size. Fold changes are represented relative to WT levels. Data represent the

mean

standard deviation (SD).

mice/group. *

; one-way Analysis

𝑁

= 4

𝑁

= 5

𝑝

< 0.05

of Variance (ANOVA) with Holm-Sidak’s multiple comparisons test. Mice were nine months

old.

Figure 3.

Mark4

knockout decreased tau phosphorylation at Ser356.

(A)

Representative

images of pSer262 tau immunostaining in the hippocampus, amygdala, and piriform cortex.

(B)

Graphs show the integrated intensity quantification normalized to the region area size. Fold

changes are represented relative to levels in PS19 mice. Data represent the mean

standard

deviation (SD).

mice/group. ns, nonsignificant; one-way Analysis of Variance

𝑁

= 4

𝑁

= 5

(ANOVA) with Dunnett’s multiple comparisons test. Insets represent the CA1, CA3, amygdala

(A), and piriform cortex (PC) regions. Scale bars, 500

μm

and 50

μm

(insets).

(C)

Representative images of pSer356 tau immunostaining with

(D)

its integrated intensity

quantification in the hippocampus, amygdala, and piriform cortex. Data represent the mean

SD.

mice/group. ns, nonsignificant; *

; **

; one-way ANOVA

𝑁

= 4

𝑁

= 5

𝑝

< 0.05

𝑝

< 0.01

with Dunnett’s multiple comparisons test. Insets represent the CA1, CA3, amygdala (A), and

piriform cortex (PC) regions. Scale bars, 500

μm

and 50

μm

(insets).

(E)

Representative images

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of total human tau (HT7) protein immunostaining with

(F)

its integrated intensity

quantification in the hippocampus, amygdala, and piriform cortex. Data represent the mean

SD.

mice/group. ns, nonsignificant; *

; one-way ANOVA with

𝑁

= 4

𝑁

= 5

𝑝

< 0.05

Dunnett’s multiple comparisons test. Insets represent the CA1, CA3, amygdala (A), and

piriform cortex (PC) regions. Scale bars, 500

μm

and 50

μm

(insets).

(G)

Immunoblot of human

total (HT7), pSer262, and pSer356 tau from brain lysates of WT, PS19, PS19:MARK4

+/-

PS19:MARK4

-/-

with

(H)

band integrated intensity quantification and normalization to

GAPDH (HT7) or total tau levels (pSer262 and pSer356). Fold changes are represented relative

to levels in PS19 samples. Data represent the mean

SD.

mice/group. ns,

𝑁

= 5

nonsignificant; *

; one-way ANOVA with Dunnett’s multiple comparisons test. See

𝑝

< 0.05

Supplementary Figure 5 for uncropped blots.

Figure 4.

Mark4

knockout reduced tau phosphorylation at AT8 sites in PS19 mice.

(A)

Representative images of AT8 (pSer202/pThr205) immunostaining, with

(B)

integrated

intensity quantification in the hippocampus, amygdala, and piriform cortex. The average

background signal from WT was subtracted. Fold changes are represented relative to levels in

PS19. Data represent the mean

standard deviation (SD).

mice/group. ns,

𝑁

= 4

𝑁

= 5

nonsignificant; *

; **

; ***

; one-way Analysis of Variance

𝑝

< 0.05

𝑝

< 0.01

𝑝

< 0.001

(ANOVA) with Dunnett’s

multiple comparisons test. Insets represent the CA1, CA3, amygdala

(A), and piriform cortex (PC) regions. Scale bars, 500

μm

and 50

μm

(insets).

(C)

Immunoblot

of AT8 and MARK4 from brain lysates of WT, PS19, PS19:MARK4

+/-

, PS19:MARK4

-/-

with

AT8 band integrated intensity quantification (brackets) and normalization to total human tau

protein levels. The average background signal from WT mice was subtracted. Fold changes are

represented relative to levels in PS19 samples. Data represent the mean

SD.

𝑁

= 5

mice/group. ns, nonsignificant; *

; one-way ANOVA with Dunnett’s multiple

𝑝

< 0.05

comparisons test. See Supplementary Figure 6 for uncropped blots.

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Page

Figure 5. Reduction of thioflavin S-positive tau aggregates and increasing tau solubility

as a result of

Mark4

knockout in PS19 mice.

(A)

Representative images of thioflavin S

staining of the hippocampus, amygdala, and piriform cortex of the mouse brain. Examples of

thioflavin S-positive (ThioS

) puncta are labeled with arrows in the magnified panels. Panels

represent CA1, CA3, amygdala (A), and piriform cortex (PC) regions. Scale bars, 500

μm

and

μm

(panels).

(B)

Quantification of the amount of ThioS

puncta in the hippocampus,

amygdala, and piriform cortex. Data represent the mean

standard deviation (SD).

𝑁

= 4

mice/group. ns, nonsignificant; *

;

Kruskal‒Wallis

test with Dunn’s multiple

𝑁

= 5

𝑝

< 0.05

comparison test.

(C)

Western blot of total human tau (HT7) from high-salt reassembly buffer

(HS-RAB), radioimmunoprecipitation assay (RIPA) buffer, and formic acid extraction of brain

lysates of WT, PS19, PS19:

Mark4

+/-

, PS19:

Mark4

-/-

mice with

(D)

band integrated intensity

quantification normalized to GAPDH levels for HS-RAB lysate. Fold changes are represented

relative to levels in PS19 samples. Data represent the mean

SD.

mice/group. No

𝑁

= 3

significant differences between groups were found (

). One-way Analysis of Variance

𝑝

> 0.05

(ANOVA) with Dunnett’s multiple comparisons test. See Supplementary Figure 7 for

uncropped blots.

Figure 6.

Mark4

knockout reduced astrogliosis

in PS19 mice.

(A)

Representative images of

Iba1 immunostaining with

(B)

quantification of the percentage of Iba1-positive area in the

hippocampus, amygdala, and piriform cortex. Data represent the mean

standard deviation

(SD).

mice/group. ns, nonsignificant (

); **

, ***

;

𝑁

= 4

𝑁

= 5

𝑝

> 0.05

𝑝

< 0.01

𝑝

< 0.001

one-way Analysis of Variance (ANOVA) with Holm-Sidak’s multiple comparisons test. Insets

represent the CA1, CA3, amygdala (A), and piriform cortex (PC) regions. Scale bars, 500

μm

and 50

μm

(insets). LSCM: laser scanning confocal microscopy images of piriform cortex.

Scale bar, 50

μm.

(C)

Representative images of GFAP immunostaining with

(D)

quantification

of the percentage of GFAP-positive area in the hippocampus, amygdala, and piriform cortex.

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Page

Data represent the mean

SD.

mice/group. ns, not significant (

); *

𝑁

= 4

𝑁

= 5

𝑝

> 0.05

; **

; one-way ANOVA with Holm-Sidak’s multiple comparisons test. Insets

𝑝

< 0.05

𝑝

< 0.01

represent the CA1, CA3, amygdala (A), and piriform cortex (PC) regions. Scale bars, 500

μm

and 50

μm

(insets). LSCM: laser scanning confocal microscopy images of piriform cortex

region. Scale bar, 50

μm.

(E)

Immunoblot of GFAP from total brain lysate of WT, PS19,

PS19:

Mark4

+/-

, PS19:

Mark4

-/-

mice with GFAP bands integral intensity quantification

normalized by GAPDH. Data are mean

SD.

mice/group. ns – non-significant, ***

𝑁

= 3

; One-way ANOVA with Holm-Sidak’s multiple comparisons test. See

𝑝

< 0.001

Supplementary Figure 8 for uncropped blots.

Figure 7.

Mark4

knockout reduced astrogliosis

in the hippocampus of wild-type (WT)

mice.

(A)

Representative images of GFAP immunostaining with

(B)

quantification of the

percentage of GFAP-covered area in the hippocampus of WT,

Mark4

+/-

, and

Mark4

-/-

mice.

Data represent the mean

standard deviation (SD).

mice/group. ns,

𝑁

= 4

𝑁

= 5

nonsignificant; **

; one-way Analysis of Variance (ANOVA) with Dunnett’s multiple

𝑝

< 0.01

comparisons test. Insets represent the CA1, CA3, amygdala (A), and piriform cortex (PC)

regions. Scale bars, 500

μm

and 50

μm

(insets). LSCM: laser scanning confocal microscopy

images of hippocampus CA1 region. Scale bar, 50

μm.

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Table 1

. Primers

Gene

Primer name

Sequence 5'

→

MARK4

common forward

GCATGTGAACCTGTGTGAAAGGAG

MARK4

wild type reverse

TCAATGATCTTAATAGCGACCTGTGG

MARK4

mutant reverse

CCCTGATACTGATCCCAGCTCCAG

P301S Tau common forward

TTGAAGTTGGGTTATCAATTTGG

P301S Tau wild type reverse

TTCTTGGAACACAAACCATTTC

P301S Tau mutant reverse

AAATTCCTCAGCAACTGTGG

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Table 2.

Antibody list

Antibody

Distributor, reference

Host

WB dilution

IHC dilution RRID

Anti-MAP2

Millipore, ab5622

Rabbit

1:500

AB_91939

Anti-PSD95

DSHB, K28/43

Mouse

1:500

AB_2877189

Anti-GAPDH

Novus, NB100-56875

Rabbit

1:3000

AB_838305

Anti-pSer262 tau

Abcam, ab131354

Rabbit

1:500

AB_11156689

Anti-pSer356 tau

Abcam, ab75603

Rabbit

1:200

AB_1310736

Anti-human tau, HT7

Thermo Fisher Scientific,
MN1000

Mouse

1:5000

1:1000

AB_2314654

Anti-MARK4

Cell Signaling Technology,
4834

Rabbit

1:250

AB_2140610

Anti-pSer202/pThr205 tau,
AT8

Thermo Fisher Scientific,
MN1020

Mouse

1:1500

1:500

AB_223647

Anti-tau, Tau5

Millipore, MAB361

Mouse

1:2000

AB_94944

Anti-GFAP

GeneTex, GTX89226

Goat

1:2000

1:500

AB_10724708

Anti-Iba1

FUJIFILM Wako, 011-27991

Goat

1:500

AB_2935833

Anti-mouse IgG

Dako, P0447

Goat

1:4000

AB_2617137

Anti-rabbit IgG

Dako, P0399

Goat

1:4000

AB_2617141

Anti-goat IgG

Dako, P0449

Rabbit

1:4000

AB_2617143

Anti-rabbit IgG, alexa fluor
488

Thermo Fisher Scientific,
A11008

Goat

1:500

AB_143165

Anti-mouse IgG, alexa
fluor 546

Thermo Fisher Scientific,
A11003

Goat

1:500

AB_2534071

Anti-goat IgG, alexa fluor
488

Abcam, ab150133

Donkey

1:500

AB_2832252

na - not applicable

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Figure 1. MARK4 knockout prolonged lifespan and rescued memory deficits in PS19 mice. (A) Transgenic

mouse breeding scheme and experimental timeline. (B) Western blot of MARK4 from total brain lysates of
WT (Mark4+/+), Mark4+/-, and Mark4-/- mice. See Supplementary Figure 1 for uncropped blots. (C) The

kinase activity of MARK4 immunoprecipitated from WT, Mark4+/-, and Mark4-/- mouse brain. The activity

was measured as counts per minute of [g-32P]ATP incorporation into substrate peptides. Data represent the

mean ± standard deviation (SD). (D)

Kaplan‒Meier

survival curve of PS19 compared with PS19:Mark4+/-

from the first generation (F1) of cross PS19 and Mark4+/-. The endpoint for survival experiments was 12

months of age: 16% of the PS19 and 65% of PS19:Mark4+/- mice survived by this time. PS19 and

PS19:Mark4+/- curves were compared using a Mantel-Cox test (***p=0.0008). N=15 (WT), N=15

(Mark4+/-), N=19 (PS19), and N=20 (PS19:Mark4+/-). (E) Total distance traveled within 10 minutes in the

open field test. Data represent the mean ± SD. N=8 to N=13 mice/group. Two-way Analysis of Variance

(ANOVA) was used to investigate the effect of the PS19 genotype (p=0.0041). *p<0.05; Tukey’smultiple

comparisons test. (F) Percentage of correct alteration in the Y-maze spontaneous alteration test. Data

represent the mean ± SD. N=8 to N=11 mice/group. Two-way ANOVA revealed the interaction effect

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between PS19 and Mark4 knockout mice (p=0.0413). *p<0.05; Tukey’s multiple comparisons test. (G) The

discrimination index was quantified to assess recognition memory in WT and PS19 mice in the presence or

absence of Mark4. Data represent the mean ± SD. N=5 to N=10 mice/group. *p<0.05; two-way ANOVA

with Tukey’s multiple comparisons test.

180x258mm (300 x 300 DPI)

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Figure 2. Mark4 knockout ameliorated the loss of synapses and dendrites in PS19 mice. (A) Representative

images of double immunostaining of the hippocampus, amygdala, and piriform cortex regions for the

postsynaptic marker PSD-95 and dendritic marker MAP2. The dashed line highlights the hippocampus (H),

piriform cortex (PC), and amygdala (A). Insets represent magnified CA1, CA3, amygdala (A), and piriform

cortex (PC) regions. Scale bars, 500

μm

and 50

μm

(insets). (B) Representative laser scanning confocal

microscopy images of neurons in the piriform cortex for PSD-95 (magenta), MAP2 (green), and DAPI (blue).

Separate channel images are in Figure 3S. Scale bar, 10

μm.(C)

Quantification of PSD-95 and MAP2

integrated intensity in the hippocampus, amygdala, and piriform cortex normalized to the region area size.

Fold changes are represented relative to WT levels. Data represent the mean ± SD. N=4 to N=5

mice/group. *p<0.05; one-way ANOVA with Holm-Sidak’s multiple comparisons test. Mice were nine

months old.

180x258mm (300 x 300 DPI)

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Figure 3. Mark4 knockout decreased tau phosphorylation at Ser356. (A) Representative images of pSer262

tau immunostaining in the hippocampus, amygdala, and piriform cortex. (B) Graphs show the integrated

intensity quantification normalized to the region area size. Fold changes are represented relative to levels in

PS19 mice. Data represent the mean ± standard deviation (SD). N=4 to N=5 mice/group. ns,

nonsignificant; one-way Analysis of Variance (ANOVA) with Dunnett’s multiple comparisons test. Insets

represent the CA1, CA3, amygdala (A), and piriform cortex (PC) regions. Scale bars, 500

μm

and 50

μm

(insets). (C) Representative images of pSer356 tau immunostaining with (D) its integrated intensity

quantification in the hippocampus, amygdala, and piriform cortex. Data represent the mean ± SD. N=4 to

N=5 mice/group. ns, nonsignificant; *p<0.05; **p<0.01; one-way ANOVA with Dunnett’s multiple

comparisons test. Insets represent the CA1, CA3, amygdala (A), and piriform cortex (PC) regions. Scale

bars, 500

μm

and 50

μm

(insets). (E) Representative images of total human tau (HT7) protein

immunostaining with (F) its integrated intensity quantification in the hippocampus, amygdala, and piriform

cortex. Data represent the mean ± SD. N=4 to N=5 mice/group. ns, nonsignificant; *p<0.05; one-way

ANOVA with Dunnett’s multiple comparisons test. Insets represent the CA1, CA3, amygdala (A), and piriform

cortex (PC) regions. Scale bars, 500

μm

and 50

μm

(insets). (G) Immunoblot of human total (HT7),

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pSer262, and pSer356 tau from brain lysates of WT, PS19, PS19:MARK4+/-, PS19:MARK4-/- with (H) band

integrated intensity quantification and normalization to GAPDH (HT7) or total tau levels (pSer262 and

pSer356). Fold changes are represented relative to levels in PS19 samples. Data represent the mean ± SD.

N=5 mice/group. ns, nonsignificant; *p<0.05; one-way ANOVA with Dunnett’s multiple comparisons test.

See Supplementary Figure 5 for uncropped blots.

180x210mm (300 x 300 DPI)

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Figure 4. Mark4 knockout reduced tau phosphorylation at AT8 sites in PS19 mice. (A) Representative images

of AT8 (pSer202/pThr205) immunostaining, with (B) integrated intensity quantification in the hippocampus,

amygdala, and piriform cortex. The average background signal from WT was subtracted. Fold changes are

represented relative to levels in PS19. Data represent the mean ± standard deviation (SD). N=4 to N=5

mice/group. ns, nonsignificant; *p<0.05; **p<0.01; ***p<0.001; one-way Analysis of Variance (ANOVA)

with Dunnett’s multiple comparisons test. Insets represent the CA1, CA3, amygdala (A), and piriform cortex

(PC) regions. Scale bars, 500

μm

and 50

μm

(insets). (C) Immunoblot of AT8 and MARK4 from brain lysates

of WT, PS19, PS19:MARK4+/-, PS19:MARK4-/- with AT8 band integrated intensity quantification (brackets)

and normalization to total human tau protein levels. The average background signal from WT mice was

subtracted. Fold changes are represented relative to levels in PS19 samples. Data represent the mean ± SD.

N=5 mice/group. ns, nonsignificant; *p<0.05; one-way ANOVA with Dunnett’s multiple comparisons test.

See Supplementary Figure 6 for uncropped blots.

180x247mm (300 x 300 DPI)

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Figure 5. Reduction of thioflavin S-positive tau aggregates and increasing tau solubility as a result of Mark4

knockout in PS19 mice. (A) Representative images of thioflavin S staining of the hippocampus, amygdala,

and piriform cortex of the mouse brain. Examples of thioflavin S-positive (ThioS+) puncta are labeled with

arrows in the magnified panels. Panels represent CA1, CA3, amygdala (A), and piriform cortex (PC) regions.

Scale bars, 500

μm

and 50

μm

(panels). (B) Quantification of the amount of ThioS+ puncta in the

hippocampus, amygdala, and piriform cortex. Data represent the mean ± SD. N=4 to N=5 mice/group. ns,

nonsignificant; *p<0.05;

Kruskal‒Wallis

test with Dunn’s multiple comparison test. (C) Western blot of total

human tau (HT7) from high-salt reassembly buffer (HS-RAB), radioimmunoprecipitation assay (RIPA) buffer,

and formic acid extraction of brain lysates of WT, PS19, PS19:Mark4+/-, PS19:Mark4-/- mice with (D) band

integrated intensity quantification normalized to GAPDH levels for HS-RAB lysate. Fold changes are

represented relative to levels in PS19 samples. Data represent the mean ± SD. N=3 mice/group. No

significant differences between groups were found (p>0.05). One-way ANOVA with Dunnett’s multiple

comparisons test. All uncropped blots are in Figure S7.

493x333mm (300 x 300 DPI)

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Figure 6. Mark4 knockout reduced astrogliosis in PS19 mice. (A) Representative images of Iba1

immunostaining with (B) quantification of the percentage of Iba1-positive area in the hippocampus,

amygdala, and piriform cortex. Data represent the mean ± standard deviation (SD). N=4 to N=5

mice/group. ns, nonsignificant (p>0.05); **p<0.01, ***p<0.001; one-way Analysis of Variance (ANOVA)

with Holm-Sidak’s multiple comparisons test. Insets represent the CA1, CA3, amygdala (A), and piriform

cortex (PC) regions. Scale bars, 500

μm

and 50

μm

(insets). LSCM: laser scanning confocal microscopy

images of piriform cortex. Scale bar, 50

μm.

quantification of the percentage of GFAP-positive area in the hippocampus, amygdala, and piriform cortex.

Data represent the mean ± SD. N=4 to N=5 mice/group. ns, not significant (p>0.05); *p<0.05; **p<0.01;

one-way ANOVA with Holm-Sidak’s multiple comparisons test. Insets represent the CA1, CA3, amygdala (A),

and piriform cortex (PC) regions. Scale bars, 500

μm

and 50

μm

(insets). LSCM: laser scanning confocal

microscopy images of piriform cortex region. Scale bar, 50

μm.

(E) Immunoblot of GFAP from total brain

lysate of WT, PS19, PS19:Mark4+/-, PS19:Mark4-/- mice with GFAP bands integral intensity quantification

normalized by GAPDH. Data are mean ± SD. N=3 mice/group. ns – non-significant, ***p<0.001; One-way

ANOVA with Holm-Sidak’s multiple comparisons test. See Supplementary Figure 8 for uncropped blots.

180x114mm (300 x 300 DPI)

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Figure 7. Mark4 knockout reduced astrogliosis in the hippocampus of wild-type (WT) mice. (A)

Representative images of GFAP immunostaining with (B) quantification of the percentage of GFAP-covered

area in the hippocampus of WT, Mark4+/-, and Mark4-/- mice. Data represent the mean ± SD. N=4 to N=5

mice/group. ns, nonsignificant; **p<0.01; one-way ANOVA with Dunnett’s multiple comparisons test. Insets

represent the CA1, CA3, amygdala (A), and piriform cortex (PC) regions. Scale bars, 500

μm

and 50

μm

(insets). LSCM: laser scanning confocal microscopy images of hippocampus CA1 region. Scale bar, 50

μm.

323x158mm (300 x 300 DPI)

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Tauopathy mouse PS19

Neuronal integrity
Memory

Lifespan

MARK4

-/-

Phospho-tau
Tau aggregates

Astrogliosis

PS19:MARK4

-/-

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