Post on 20-Jan-2021
transcript
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