Introduction

Conclusions

• In this study, we have early evidence that pSer208 is involved in

enhancing tau aggregation in cell culture and is a major component

of human pathology in tauopathies such as AD, PSP, and CBD.

• Compared to antibodies against AT8, antibodies against pSer208

are more specific for mature tangles and neuronal pathology. This

suggests astrocytic pathology in CBD and PSP may have lower

amounts of pSer208.

• This supports the hypothesis that different tau strains may also have

different phosphorylation patterns.

• In the future, a specific antibody against pSer208 may be useful for

both diagnosis and immunotherapy.

References & Funding

This work was supported by grants (7AZ25 and 9AZ17) from Florida

Department of Health.

Tau Phosphorylation of Ser208 Promotes Aggregation of Wild Type tau in Alzheimer’s disease and Other TauopathiesYuxing Xia1,2, Stefan Prokop1,2, Justin Kim1,2, Zachary Sorrentino1,2 , Kim Gorion1,2, Brach Bell1,2, Alyssa Manaois1,2, Kevin Strang1,2, Benoit Giasson1,2

1Center for Translational Research in Neurodegenerative Disease, 2Department of Neuroscience, University of Florida College of Medicine

Contact: yuxingxia@ufl.edu

Results• Tau is a microtubule-associated protein that normally stabilizes

microtubules and promotes tubulin assembly.

• In Alzheimer’s disease (AD) and other tauopathies, tau becomes

hyperphosphorylated and aggregates to form neurofibrillary tangles, which

directly correlates with clinical symptoms.

• Antibodies against hyperphosphorylated tau such as the AT8 epitope

(pS199/ps202/pT205) are used for clinical staging and diagnosis of AD.

• We show evidence that a new phosphorylation site pSer208 promotes

aggregation of wild type tau and have created a new monoclonal antibody

specific for pSer208, which will be useful for diagnosis and therapy.

Experimental Design

Results

• Cell culture: HEK293T cells were maintained in 10% FBS with DMEM.

Different plasmids were transfected by calcium phosphate precipitation.

• Western blot, antibodies, and quantification: 3026 antibody for total tau,

tubulin antibody for control. Western blots were quantified by ImageJ.

• ELISAs were performed using plates coated with different tau peptides.

• Standard immunohistochemistry was performed on human tissue

provided by the UF Neuromedicine brain bank.

Figure 1: Phosphomimetic

of S202E, T205E, & S208E

promotes self-aggregation

of wild type tau in cell

culture. Wild type tau does

not normally aggregate

even when seeded by K18.

P301L is a common tau

mutation that aggregates

with seeding. S202E and

T205E phosphomutant

does not aggregate until it

is enhanced by S208E.

Figure 2: Different antibodies near the AT8 epitope

were compared using ELISA. We validated our new

monoclonal to be specific for pSer208.

Figure 5: Semi-quantitative counting of different inclusions in patients with Alzheimer’s

disease. Different 20X fields were selected in the hippocampus regions of 6 different AD

patients. Pre-tangles, mature tangles, and neuritic plaques were counted in each area and

plotted above. Antibodies against pSer208 is more specific for mature tangles. Similarly,

pSer208 may be less present in neuritic plaques.

Figure 4: Antibodies against pSer208 stain neuronal and oligodendrocytic pathology

better than astrocytic pathology in patients with progressive supranuclear palsy

(PSP) and corticobasal degeneration (CBD). AT8 antibodies stains (A) astrocytic

plaques in CBD and (B) tufted astrocytes, (C) coiled bodies, and (D) globose tangles

in PSP. pSer208 may be an epitope that is less present in astrocytic pathology such

as astrocytic plaques in CBD and tufted astrocytes in PSP.

Figure 3: AT8-related and pSer208 antibodies stain different types of pathology in patients

with Alzheimer’s disease. Antibodies against the AT8 epitope such as AT8, CP13, and 7F2

strongly stain (A) neurofibrillary tangles, (B) neuritic plaques, and (C) neuropil threads.

However, antibodies against pSer208 seem to be more specific for mature tangles.

2D

1

CP

13

7F

2

3G

12

AT

8

PB

S

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

B in d in g S p e c if ic ity o f A T 8 A n t ib o d ie s

A n tib o d ie s

Op

tic

al

De

ns

ity

(O

D)

p S 1 9 9 p e p tid e

p S 2 0 2 p e p tid e

p T 2 0 5 p e p tid e

S 2 0 8 p e p tid e

p S 2 0 8 p e p tid e

p S 1 9 9 /p S 2 0 2 /p T 2 0 5 (A T 8 )

p e p tid e

AT

8

pS

208

0

2 5

5 0

7 5

1 0 0

P e r c e n t o f M a tu re T a n g le s

in H ip p o c a m p u s o f A D P a tie n ts

Ra

tio

of

Ma

ture

Ta

ng

les

to T

au

P

os

itiv

e N

eu

ro

ns

* * * *

AT

8

pS

208

0

2

4

6

N e u r it ic P la q u e s in th e

H ip p o c a m p u s o f A D P a t ie n ts

Nu

mb

er o

f N

eu

rit

ic P

laq

ue

s

* * * *

Summary of ELISA results:

• AT8 – pS199, pS202, pT205

• CP13 – pS202

• 7F2 – pT205

• 3G12 – pS208

• 2D1 – not phospho-dependent