neuronal antibody uptake and tau clearance

Neuronal Antibody Uptake and Tau Clearance

1

Antibody Uptake into Neurons Occurs Primarily via Clathrin Dependent Fc Receptor Endocytosis, and

is a Prerequisite for Acute Tau Clearance*

Erin E. Congdon1, Jiaping

Gu

1, Hameetha B. R. Sait

1, Einar M. Sigurdsson

1,2*

1 Department of Neuroscience and Physiology,

2 Department of Psychiatry, New York University School

of Medicine, New York, NY 10016

*Running title: Fc Receptor Mediated Antibody Uptake

To whom correspondence should be addressed: Einar M. Sigurdsson, Department of Neuroscience and

Physiology, and Department of Psychiatry, New York University School of Medicine, 550 First Ave.

MSB 459, New York, NY 10016 USA. Tel: (212)263-3913 Fax: (212)263-2160 email:

[email protected]

Keywords: Alzheimer’s disease, antibodies, endocytosis, immunotherapy, tau

____________________________________________________________________________________

Background: How tau immunotherapy clears tau

is not well known.

Results: Tau antibody uptake correlates with tau

levels, is primarily via clathrin-dependent FcγII/III

endocytosis, and is required for acute tau

clearance.

Conclusion: Tau antibodies are mainly neuronal

co-localized with tau aggregates, following

receptor-mediated uptake, promoting tau

clearance.

Significance: Results support intracellular

clearance as a viable route for immunotherapy,

and have major implications for future

development.

________________________________________

Tau antibody therapy is effective in transgenic

mice but the mechanisms of tau clearance are

not well known. To this end, tau antibody

uptake was analyzed in brain slice cultures and

primary neurons. Internalization was rapid

(<1h), saturable, and substantial compared to

control mouse IgG. Furthermore, temperature

reduction to 4oC, excess of unlabeled mouse

IgG, or excess tau antibodies reduced uptake in

slices by 63%, 41%, and 62% respectively

(p=0.002, 0.04, 0.005). Uptake strongly

correlated with total and insoluble tau levels

(r2=0.77 and 0.87, p=0.002 and 0.0002),

suggesting that tau aggregates influence

antibody internalization and/or retention

within neurons. Inhibiting phagocytosis did not

reduce uptake in slices or neuronal cultures,

indicating limited microglial involvement. In

contrast, clathrin specific inhibitors reduced

uptake in neurons (≤ 78%, p<0.0001) and slices

(≤ 35%, p=0.03), demonstrating receptor-

mediated endocytosis as the primary uptake

pathway. Fluid phase endocytosis accounted for

the remainder of antibody uptake in primary

neurons, based on co-staining with internalized

dextran. The receptor-mediated uptake is to

large extent via low affinity FcγII/III receptors,

and can be blocked in slices (43%, p=0.04) and

neurons (53%, p=0.008) with an antibody

against these receptors. Importantly, antibody

internalization appears to be necessary for tau

reduction in primary neurons. Overall, these

findings clarify that tau antibody uptake is

primarily receptor-mediated, that these

antibodies are mainly found in neurons with

tau aggregates, and that their intracellular

interaction leads to clearance of tau pathology,

all of which have major implications for

therapeutic development of this approach.

________________________________________

A host of neurodegenerative diseases involve

deposition of abnormal protein aggregates in the

http://www.jbc.org/cgi/doi/10.1074/jbc.M113.491001The latest version is at JBC Papers in Press. Published on October 25, 2013 as Manuscript M113.491001

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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brain including Alzheimer’s, Parkinson’s,

Huntington’s, and prion diseases. In Alzheimer’s

disease (AD), the defining lesions are amyloid-β

plaques and neurofibrillary tangles (NFT)

composed of the microtubule-associated protein

tau. Active and passive immunotherapies are

among the more attractive strategies for clearing

these pathological proteins. Immunotherapy was

first utilized in the context of AD to clear Aβ

plaques and improve cognitive function in animal

models (1-6). However, early clinical trials were

suspended over concerns regarding encephalitis in

a small percentage of participants (7). Although

immunization was effective at reducing amyloid

burden, there was no beneficial effect on synapses

or clinical progression (8). Thus, plaque clearance

alone was insufficient to alter disease course.

Following further refinement, additional clinical

trials targeting various forms of Aβ are ongoing.

Recent findings from Phase III trials of

Bapineuzumab and Solanezumab, antibodies

recognizing amyloid-β, have been disappointing.

No overall cognitive benefits were seen with either

antibody, although subgroups showed significant

albeit minimal slowing of memory deterioration in

mild AD with Solanezumab (9).

Besides Aβ, tau lesions present an

attractive target for disease modifying

interventions. Our group initially demonstrated the

feasibility of targeting these aggregates by

effectively utilizing active and passive tau

immunization in transgenic (Tg) models of

tauopathy. Both approaches targeted the phospho-

serine 396 and 404 region and consistently

reduced pathological tau and ameliorated related

functional impairments (10-12). These findings

have been confirmed and extended by other

groups as well (13-18).

Recent results have shown that tau

antibodies not only co-localize with intraneuronal

pathological tau, but also with

endosomal/lysosomal pathway markers (10,19).

Similar findings have been observed by others

utilizing antibodies for α-synuclein and Aβ (20-

22). However, the mechanism by which antibodies

enter cells remained unknown. To clarify this

issue, we studied uptake of labeled tau antibodies

in slice cultures prepared from Tg, and wild type

mice, as well as in primary neurons prepared from

Tg mice. The antibody used in these studies,

4E6G7, was developed by our group and has

previously showed promising results in

preliminary experiments in transgenic animals

(23). Mechanism of uptake was assessed via

temperature manipulation, blocking with excess

IgG, anti-tau, or anti-Fc antibodies, and

pharmacological interventions. In addition, the

effect of antibody treatment on tau levels in

primary neuronal cultures was assessed. Our

findings indicate that uptake of tau antibodies is an

energetic process, and to a large extent occurs

through a clathrin-mediated endocytic pathway via

FcγII/III receptors. Importantly, antibody

internalization is necessary for clearance of tau in

neuronal cultures.

Experimental Procedures

Materials

Radioactive iodine was purchased from Perkin

Elmer (Waltham, MA) and Pierce Iodobeads

(Pierce, Rockford, IL), iodination reagent and

desalting columns from Fisher Scientific

(Waltham, MA). Chlorpromazine, cytochalasin D,

filipin III, dansylcadaverine, Superblock, and

SX1-4 scintillation liquid were also purchased

from Fisher Scientific. Neurobasal A, MEM,

Glutamax, sodium pyruvate, glutamine, HBSS,

HEPES, DNAse, and B27 vitamins were

purchased from Invitrogen (Grand Island, NY).

Anti Fcγ II/III (CD16/CD32) and Fcγ I (both rat

anti mouse) were obtained from BD Biosciences

(Franklin Lakes, NJ), mouse IgG1κ from

eBioscience (San Diego, CA), PHF-1 (mouse

monoclonal) antibody was a gift from Dr. Peter

Davies. Pan tau rabbit polyclonal antibody

corresponding to residues 243-441 was purchased

from Dako (Carpinteria, CA). Rabbit polyclonal

anti tau pSer199

antibody was purchased from

Santa Cruz Biotechnology (Dallas, TX). Complete

protease inhibitor cocktail tablets were purchased

from Roche (San Francisco, CA).

Animals

Animals utilized for slice cultures in experiments

with I125

labeled antibodies were aged adults (15-

18 months) from three different mouse lines. The

htau/PS1 (12) line was utilized for the majority of

the experiments. These animals express all six

isoforms of human tau in the absence of murine

tau (24). In addition, they express human


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presenilin containing the M146L mutation (25).

For experiments correlating pathology with

antibody uptake, additional aged animals from the

3xTg line (26) and their wild type littermates were

used. For experiments with fluorescently labeled

antibodies, adult animals (3-4 months) from the

JNPL3 (27) line were used. These animals express

human 0N4R tau containing the P301L mutation.

JNPL3 pups at postnatal day 0 were utilized for

primary neuronal cultures. In all lines, animals of

both sexes were enrolled. All animals were housed

in accordance with IACUC regulations in

AAALAC approved facilities, and given free

access to food and water ad libitum.

4E6G7 preparation

Monoclonal antibodies were generated by

GenScript Inc. Wild-type (WT) BALB/c mice

were immunized with a peptide corresponding to

the phospho-serine396/404

region

(cTDHGAEIVYK[pSer]PVVSGDT[pSer]PRHL).

The peptide was conjugated to KLH via the

cysteine residue and mice showing a satisfactory

immune response were used in hybridoma

production. Monoclonal antibody 4E6G7 was

selected and purified from the culture supernatant

with a low EU protein A affinity column.

I125

labeling

Uptake studies utilized 4E6G7, an IgG1κ

isotype monoclonal antibody developed by this

lab. 4E6G7 was selected from a panel of

antibodies made by subcontractor Genscript Inc.

(Piscataway, NJ) against a phospho-epitope

encompassing serine 396 and 404 as detailed

above. This antibody selectively recognizes this

region, primarily the phospho-serine 404, with

lesser reactivity towards non-phospho tau. 4E6G7

and control IgG1κ were labeled with carrier free

Na125

I using Pierce Iodination beads and reagents

according to manufacturer’s instructions. Specific

activity was determined as 2.04 and 2.12 μCi/μg

respectively.

Fluorescent labeling

4E6G7 was labeled using the Alexa Fluor

568 Labeling Kit from Invitrogen. Briefly, the

antibody was incubated with reactive dye at room

temperature for one hour with stirring. The elution

column was prepared as per the instructions and

the antibody dye mix was added, followed by

antibody collection and verification of labeling.

Slice cultures Slice cultures were prepared as previously

described (28). Briefly, mice were killed via

cervical dislocation and the brain removed. The

brainstem and cerebellum were discarded and the

two hemispheres separated. Each hemisphere was

cut into 400 μm sections on a Brinkman tissue

chopper. Slices were separated in ice cold culture

buffer (62.3 mM NaCl, 1.5 mM KCl, 0.62 mM

KH4PO4, 4.01 mM MgSO4, 1.35 mM CaCl2, 1.74

mM NaHCO3, 5 mM Glucose, 1 mM Ascorbic

acid, 0.02 mM ATP) and distributed among six

wells. Slices were left for 30 minutes at room

temperature to recover. Following recovery, slices

were placed into a Beion BS3 brain slice chamber

with oxygenated culture buffer.

Each apparatus contains six wells

allowing each animal to be utilized for multiple

conditions as well as serve as its own internal

control. Because pathology is regional, slices are

distributed among the wells such that each well

contains a similar selection of brain regions.

Primary neuronal cultures

Neuronal cultures were prepared from

JNPL3 pups at postnatal day 0. Briefly, plates

were coated for three hours with Poly-L-lysine.

Brains were harvested, meninges and brainstem

were removed. The remaining brain tissue was

washed five times in HBSS+++ (975 ml Hank’s

Balanced Salt Solution, 10 ml 1M HEPES, 5 ml

pen/strep, 10 ml 100 mM Sodium Pyruvate) and

then incubated with 200 μl of 0.5% trypsin for 15

minutes. Trypsin was neutralized with an equal

volume of plating media (423.5 ml MEM, 15 ml

Glutamax (100x), 5 ml 200 mM Glutamine, 50 ml

FBS, 4 ml B27, 2.5 ml pen/strep) and 100 μg

DNAse added to further dissociate the cells.

Tissue was again washed five times with

HBSS+++, and centrifuged for one minute at 0.5 x

g. Tissue was then resuspended in 1 ml plating

media and mechanically dissociated to form a cell

suspension. Cells were centrifuged again and the

resuspended cells were evenly distributed among

the wells. Plating media was replaced with culture

media (499 ml Neurobasal A, 1 ml B27

supplement, 17 μl BME) the following day. Cells


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were maintained in culture and allowed to develop

processes prior to use in experiments.

Antibody uptake in primary neurons and brain

slices

Cells were prepared as described on glass

coverslips. Fluorescently labeled 4E6G7 was

added to the culture media and cells incubated at

37oC for one hour. Cells were then washed and

fixed for 10 minutes. Coverslips were washed

again and mounted on slides using Dako

Cytomation fluorescent mounting media. Images

were collected on a Nikon Eclipse Ti confocal

microscope. Additional cells were incubated with

fluorescently labeled dextran as well as 4E6G7 for

one hour at 37oC.

Brain slices were incubated at 37oC in the

presence of fluorescently labeled 4E6G7 for two

hours. Slices were then fixed, sectioned and

stained with anti-tau antibodies. Sections were

mounted using Dako Cytomation fluorescent

mounting media. Images were collected using a

Zeiss 700 confocal microscope.

Staining of primary neurons and brain slices

Cells were cultured as described, washed

and fixed for 10 minutes. Cells were

permeabilized with 0.3% Tween in PBS for an

additional 10 minutes. Coverslips were incubated

with primary antibody at room temperature for

two hours. Following primary antibody, coverslips

were washed three times, and then incubated with

secondary antibodies for one hour. Coverslips

were washed an additional three times and then

mounted on slides.

Sections from acute brain slice cultures

were also utilized for staining. Sections were fixed

and permeabilized with 0.3% Tween in PBS.

Slices were incubated in primary antibodies

overnight and secondary for one hour at room

temperature.

Dose response and time course experiments

Brain sections prepared as described

above from htau/PS1 mice (n = 3, age 15-18

months) were incubated with increasing

concentrations of I125

-labeled 4E6G7 antibody.

Concentrations of antibody in culture buffer

ranged from 0.01 μg to 5 μg per ml. Each brain

was sectioned at 400 μm and sections were

divided evenly between the treatment groups.

Sections were maintained in oxygenated

buffer at 37oC. At 30, 60 and 120 minutes,

sections were removed, weighed and rinsed with

acidified culture buffer (pH 5). Sections were

washed a further three times in ice cold culture

buffer to remove surface bound antibodies.

Following rinsing, sections were placed in plastic

vials with 5 ml scintillation liquid, and

radioactivity was measured on a Beckman LS

6500 liquid scintillation counter. CPM values were

converted to nCi and corrected for differing tissue

mass. Average and standard error of the mean

(SEM) were determined from the results, plotted

against the concentration of antibody in buffer,

and fit to a linear curve.

To determine whether uptake was

saturable, an additional group of brain slices from

htau/PS1 animals were incubated with increasing


-labeled 4E6G7 with an

additional 10 μg/ml dose added. For this

experiment, all samples were collected after 60

minutes of incubation. Samples were prepared and

radioactivity determined as described above. A

saturation curve was also prepared using slices

from wild type animals. All samples were

collected after 60 minutes incubation with up to 10

μg/ml I125

labeled 4E6G7.

Further dose response experiments were

carried out utilizing WT animals and mice from

the 3xTg line (n = 3 per group). Brain sections

were prepared as described and incubated for 1

hour at 37oC with increasing concentrations of I

125

labeled 4E6G7 (0.01-5 μg). Sections were washed

and radioactivity measured. As before, values

were converted and plotted against the

concentration of antibody in the incubation buffer.

With fluorescently labeled 4E6G7,

antibody incubation and washing were carried out

using sections from JNPL3 mice (n = 3 per group,

age 3-4 months) exactly as with radiolabeled

antibodies. Following washing, slices were

homogenized and centrifuged for 15 min and the

values were corrected for total protein

concentration in the samples. A standard curve

was generated by adding antibodies to brain

homogenate with a known protein concentration.

Temperature sensitivity

Prior to experiments, the slice chamber


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was placed at 4oC for 2 hours. Sections from

htau/PS1 mice (n = 3) were prepared as described

and incubated at 4oC for one hour in culture buffer

containing 0.5 μg/ml of 4E6G7 antibody.

Following treatment, slices were weighed, washed,

and radioactivity measured. Results were

compared with those obtained from 37oC control

sections. As before, fluorescence experiments

were carried out using the same procedure. For

each group average and SEM were determined.

Two tailed t-test was used to determine

significance.

Effects of excess IgG and 4E6G7

In addition to temperature, the effects of

an excess of mouse IgG on uptake was examined.

For each animal (n = 3 htau/PS1 mice), the brain

sections were separated into three groups, control

without antibody, 0.5 μg/ml 4E6G7, and 0.5 μg/ml

4E6G7 plus an excess of unlabeled mouse IgG1κ

(either 5 or 50 μg/ml). Samples were collected and

processed as described above. The effect of excess

4E6G7 was tested using the same procedure.

Samples were incubated with 0.5 μg/ml I125

labeled 4E6G7 alone, or I125

labeled 4E6G7 in the

presence of 10x or 100x excess unlabeled 4E6G7.

Averages and SEM were determined for each

group. Fluorescence experiments were done with

an additional group of JNPL3 mice (n = 3, age 3-4

months). As with temperature experiments, a two

tailed t-test was used to determine significant

differences between groups.

Endocytosis inhibitors Slice cultures were prepared from

htau/PS1 mice as described above. In order to

understand the contribution of different types of

endocytosis, three different inhibitors were

utilized, filipin III (caveolin), chlorpromazine

(clathrin), and cytochalasin D (phagocytosis). As

with other experiments, slices were incubated with

0.5 μg/ml 4E6G7 for one hour at 37oC. Increasing

concentrations of inhibitors were added to each

sample and radioactivity determined. The same

protocol was utilized to assess the effect of the

drugs on primary neuronal cultures; with the

exception that chlorpromazine was replaced with

dansylcadaverine due to solubility issues in the

cell culture media. Cells were washed in acidified

HBSS+++ (pH 5), neutral HBSS+++ and lysed

with RIPA. As with slices, final radioactivity was

corrected for protein concentration. For both slices

and neurons, a one way ANOVA followed by

Tukey’s LSD was utilized to determine

significance.

Fc block

To determine the contribution of Fc

receptors to antibody uptake, slices were prepared

as described above (htau/PS1 mice, n = 3), and

incubated at 37oC for one hour with 0.5 μg/ml

4E6G7. Increasing concentrations of anti

CD16/CD32 Fc receptor blocking antibody (Fc

Block anti CD16/CD32 (BD Bioscience) 0.05

μg/ml – 50 μg/ml) were added to the culture

buffer. Following incubation, slices were treated

as described and average radioactivity per

treatment group determined. Additional slices

were incubated with increasing concentrations of

an antibody against FcγI receptor (anti-FcγRI,

0.05-50 μg/ml).

Fc Block was also added to primary

neuronal cultures prepared from JNPL3 pups.

Cells were incubated with 0.5 μg/ml 4E6G7 and

increasing concentrations (0.5-10 μg/ml) of Fc

Block for 1 hour at 37oC. Cells were washed

following treatment and processed as described. A

second group of neurons was treated with anti-

FcγRI (0.5-10 μg/ml) under the same conditions.

Final radioactivity for all samples was obtained as

described and normalized for total protein

concentration. Significance in both slices and

neurons was determined using a one way ANOVA

followed by Tukey’s LSD.

Effects of 4E6G7 on Tau

Primary neurons were prepared as

described above to determine the effect of 4E6G7

on intraneuronal tau levels. Cells were incubated

for 24 or 72 hours in the presence of 4E6G7 alone

(1, 10, or 20 μg/ml) or 4E6G7 and 1μg/ml

dansylcadaverine. After treatment, cells were

collected and processed for immunoblotting.

Immunoblots for total tau levels, tau

phosphorylated at ser199

, and actin were carried

out. Data was analyzed using a two way ANOVA

and Bonferroni post hoc test.

Immunoblotting

Brain sections not used in uptake


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experiments and primary neurons were retained

for immunoblotting. Each sample was

homogenized in modified RIPA buffer (50 mM

Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1

mM NaF, 1 mM Na3VO4, 1 μg/ml complete

protease inhibitor cocktail (Roche)) and subjected

to a low speed spin (22 x g) to remove the

membrane fraction. The supernatant was retained

and volumes adjusted for total protein

concentration. Samples were diluted in O+ buffer

(62.5 mM Tris-HCl, pH 6.8, 5% glycerol, 2-

mercaptoethanol, 2.3% SDS, 1 mM EGTA, 1 mM

phenylmethylsulfonyl fluoride, 1 mM NaVO4, 1

μg/ml Roche complete protease inhibitor cocktail)

and boiled for 5 min and loaded onto a 12%

polyacrylamide gel. Gels were transferred at 100V

for one hour and blots incubated overnight in

appropriate dilutions of primary antibody. Blots

were washed and incubated with a peroxidase

labeled mouse secondary antibody for one hour

followed by further washing.

Blots were developed using a Fuji LAS-

4000 and signal quantified with Multigauge.

Chemiluminescent signal was measured, and when

appropriate plotted against radioactivity, and fit to

a linear curve.

RESULTS

4E6G7 recognizes pathological tau in tissue

In sections from 3xTg and WT animals

aged 22 months, 4E6G7 shows strong neuronal

staining in the hippocampus of 3xTg mice (Figure

1A). PHF-1 also produced robust staining (Figure

1B) in transgenic animals, although the pattern is

less punctuate. However, 4E6G7 did not stain

tissue from WT mice of the same strain

background (Figure 1C). This region has

prominent tau and Aβ lesions in this model.

Uptake is rapid and saturable

Slice cultures from htau/PS1 transgenic

mice (n = 3, 15-18 months) were incubated at

37oC with increasing concentrations (0-10 μg/ml)

of I125

labeled tau antibody 4E6G7 raised against

the pSer396/404

region of the tau protein. Individual

sections were removed at 30, 60 and 120 minutes.

Final intracellular radioactivity at all endpoints

was linear up to antibody concentrations of 5

μg/ml at one hour incubation (Figure 2A), with

saturation occurring between 5 and 10 μg/ml

(Figure 2B). Similar values were obtained when

utilizing fluorescent antibodies with JNPL3 slices,

with uptake reaching saturation between 5 and 10

μg/ml (Figure 2C). This pattern appears fairly

robust across mouse lines, as similar results were

obtained in wild type animals using I125

labeled

4E6G7 at one hour incubation (Figure 2D). In

addition, uptake was rapid and remained steady at

each time point. On the basis of these results, other

experiments were carried out using a 0.5 μg/ml

antibody concentration and an incubation time of

one hour. In control experiments we have found

that slice cultures prepared from wild type animals

show reactivity to PHF-1 on Western blots. It

appears that the degree of PHF-1 reactivity is

increased due to the process by which the slice

cultures are made. When compared to sections

allowed to recover at room temperature, PHF-1

chemiluminescent signal in flash frozen

hemispheres was reduced by 28% (Figure 3).

To validate the system we performed

several control experiments. Slices that had been

incubated with labeled 4E6G7 were further

sectioned at 30 μm. Each section was added to 5

ml scintillation fluid and radioactivity measured.

There was no significant difference in

radioactivity between sections taken at different

levels (data not shown) indicating homogeneous

distribution of antibody in tissue. Additionally, we

compared sections with unsectioned hippocampus

in order to determine the contribution of broken

cell membranes to final radioactivity. There was a

30% decrease in radioactivity in unsectioned

tissue, indicating that at least a portion of antibody

uptake is via damaged membranes.

Uptake is reduced at low temperatures

To ascertain the effect of temperature on

antibody uptake, the slice chamber was chilled for

two hours at 4oC prior to the addition of brain

slices. Slices were prepared as described above

and incubated at 4oC for one hour with 0.5 μg/ml

of I125

labeled 4E6G7. Samples were collected,

weighed, rinsed and levels of radioactivity

determined. Lowered temperatures significantly

reduced the uptake of antibody from 0.171 ± 0.6 to

0.031 ± 0.006 nCi/mg (p = 0.002; 63% decrease

relative to 37oC control), suggesting that

antibodies are internalized mainly via an energy

dependent process. The same pattern of uptake


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was observed in JNPL3 slices incubated with

fluorescently labeled 4E6G7 with 4oC sections

showing a 35% reduction in signal 0.048 ± .009 to

0.031 ± 0.004 µg antibody/ mg tissue (p = 0.05).

Excess IgG and unlabeled 4E6G7 partially

block antibody uptake

In addition to temperature, co-incubation

with an excess of mouse IgG1κ also reduced

uptake of 4E6G7. Slices from each animal were

separated into three groups, control, 0.5 μg/ml

4E6G7 and 0.5 μg/ml 4E6G7 plus IgG1κ. The

presence of a 10x excess of mouse IgG1κ resulted

in a 41% decrease in radioactivity, a significant

reduction relative to antibody alone (p = 0.04,

Figure 4A). A second group of slices was prepared

the same way and incubated with 100x excess

IgG1κ. Again radioactivity was significantly

reduced relative to 0.5 μg/ml 4E6G7 alone (40%

decrease, p = 0.02, Figure 4A), but the additional

IgG1κ did not lead to further inhibition of uptake

(Figure 4A). Excess IgG also resulted in a 29%

decrease in uptake when using fluorescently

labeled antibodies (p = 0.05; Figure 4B).

Additional slices were incubated with 0.5 μg/ml

I125

labeled 4E6G7 and either 5 or 50 μg/ml non-

radiolabeled 4E6G7. Addition of a ten-and

hundred-fold excess of 4E6G7 reduced labeled

antibody uptake by 35% and 62%, respectively (p

= 0.01 and 0.005; Figure 4C). Both the mouse IgG

and unlabeled 4E6G7 compete with I125

-4E6G7 for

binding to the receptors on the cell surface.

However, the apparently greater effect of 4E6G7

at the 100X dose (62% vs. 40% reduction in

radioactivity) may be related to its effect on

retention. Unlike the mouse IgG, the unlabeled

4E6G7 can compete with I125

-4E6G7 for binding

with intracellular tau. Thus the excess of 4E6G7

may affect both uptake and retention of the labeled

antibody. In control experiments, we found that as

much as 30% of the final radioactivity is the result

of antibody uptake through damaged cells. Thus,

this may mask the true extent of blockage, and

prevent complete blockage of uptake.

Pathology correlates with antibody uptake

Slice cultures were created from animals

of three different mouse lines (htau/PS1, 3xTg,

and wild type (WT), n = 3 animals per group) with

differing levels of tau. Brain slices from each

animal were incubated at 37oC for one hour with

increasing concentrations of I125

labeled 4E6G7

antibody. Average radioactivity was measured and

nCi/mg of tissue determined. In addition, brain

sections from each animal were frozen and

retained for tau immunoblotting.

Animals from the htau/PS1 line showed

significantly higher levels of total tau and stronger

PHF-1 immunoreactive bands in the sarkosyl

insoluble fraction relative to both 3xTg and WT

animals (Figure 5A-D). Both transgenic lines

(htau/PS1 and 3xTg) expressing tau had

significantly higher levels of radioactivity relative

to WT mice at 1 µg/ml (49% and 34%,

respectively; p = 0.002 and 0.04) and 5 μg/ml

4E6G7 (86% and 30% respectively; p= 0.008 and

0.02). Further, at the 5 μg/ml dose, animals from

the htau/PS1 line had significantly higher levels of

uptake than the 3xTg line (43% increase, p = 0.02;

Figure 5E).

Within the identified mouse groups, levels

of both total tau and sarkosyl insoluble tau

correlated highly with final radioactivity (r2 0.77

and 0.87, p = 0.002 and 0.0002 respectively;

Figure 5F, G) for pooled samples. These results

indicate that higher levels of intracellular tau and

degree of phosphorylation at Ser396/404

may

promote greater uptake or retention of the 4E6G7

antibody in the tissue. As stated above, the method

used to make the slices produces an increase

(28%) in tau phosphorylated at Ser396/404

in WT

animals (Figure 3). This may account for the

higher than expected degree of 4E6G7

uptake/retention in the WT mice.

IgG1κ, the same isotype as 4E6G7, was

also labeled with I125

and incubated with sections

from htau/PS1 and WT animals as described. As

with 4E6G7, htau/PS1 animals had higher levels

of final radioactivity at 0.5, 1 and 5 μg/ml IgG

concentrations (71%, 73% and 48% increase, p =

0.003, 0.025, and 0.020, respectively relative to

WT mice). In both cases, however, the uptake was

greatly reduced relative to 4E6G7 treated htau/PS1

animals at 1 and 5 μg/ml IgG concentrations (84%

and 91% decrease, p = 0.008 and 0.00007) (Figure

5H). The difference in the overall levels of IgG1κ

uptake relative to 4E6G7 may be explained by

retention of the antibody in the tissue. 4E6G7 will

be bound to tau and thus less subject to recycling

out of the cell than the unbound IgG. Additionally,


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excess IgG may be degraded more rapidly by the

lysosomal system.

Antibody uptake occurs mainly via clathrin

mediated endocytosis

Endocytosis inhibitors were utilized to

further clarify the mechanism of antibody uptake.

Brain slices from htau/PS1 mice were incubated

with varying concentrations of either

chlorpromazine, filipin III, or cytochalasin D.

Phagocytosis inhibitor cytochalasin D did not

significantly alter antibody uptake (Figure 6A).

Filipin III did not affect final radioactivity at any

of the concentrations used, although the 50 µg/ml

dose produced a trend towards reduced uptake (p =

0.07), indicating caveolin mediated endocytosis

may be involved but is not a major route of uptake

(Figure 6A). Chlorpromazine produced a

significant reduction in antibody uptake (p = 0.03)

and at the 20, 50, and 100 μg/ml doses, final

radioactivity was reduced to 85%, 66%, and 66%

of control values, respectively (Figure 6A). These

results indicate that uptake is occurring to a large

extent via clathrin dependent endocytosis.

Primary neuronal cultures prepared from

JNPL3 mice were also treated with the

endocytosis inhibitors utilized above with

chlorpromazine replaced by dansylcadaverine as

the former was not completely soluble in cell

culture media. In each case, the cells were

incubated with inhibitors for one hour at 37oC in

culture media containing 0.5 μg/ml I125

labeled

4E6G7 and increasing concentrations (0.5 – 50

μg/ml) of drug (Figure 6B). Addition of

dansylcadaverine produced significant reductions

in final radioactivity at every dose used (p =

0.00006 - 0.000005) and up to 78% reduction in

signal relative to control. In contrast, filipin III

produced a significant decrease in radioactivity at

only the highest dose (p = 0.04, 30% reduction).

Cytochalasin D had no significant effect. These

data indicate that the majority of antibody uptake

in neurons occurs via clathrin mediated

endocytosis, but that a smaller fraction is

internalized by caveolin dependent pathways.

Other mechanisms may contribute fractionally to

antibody uptake. Data is less clear in the slice

cultures, where multiple cells types are present and

drugs may not have fully penetrated the tissue.

Longer incubation times with the inhibitors, or

higher concentrations, may be necessary to

observe full effect in that model.

To ensure that observed uptake is receptor

mediated, as hypothesized, rather than fluid phase

endocytosis, JNPL3 primary neurons were

incubated with both 4E6G7 and fluorescently

labeled dextran for one hour at 37oC. Images were

collected and merged (Figure 7 A-C). The

numbers of red, green and co-localized puncta

were determined (~1700 puncta counted in total).

Only 17 ± 1.2 % of the total puncta counted were

positive for both dextran and 4E6G7. Of those

puncta positive for 4E6G7, 27 ± 1.4% were

yellow, indicating that about one fourth of

antibody uptake occurs via fluid phase endocytosis

(Figure 8D).

Incubation with FcγRII/III antibody, but not

FcγRI reduces 4E6G7 uptake

Primary neurons cultured from JNPL3

mice were stained with Dako pan tau polyclonal

antibody, and FcγII/III antibody (Figure 8 A-C).

Images show that the receptors are distributed

throughout the cells. Additionally, sections from

JNPL3 mice were also stained with total tau and

FcγII/III antibodies showing a similar pattern to

that seen in the primary neurons (Figure 8 D-F).

To visualize antibody uptake, additional cultured

neurons were incubated with fluorescently labeled

4E6G7 for one hour. Images show that the

majority of the 4E6G7 antibody is visible as bright

puncta bound to tau within the neurons (Figure 8

G-I). Similar results were seen in sections from

JNPL3 mice incubated with fluorescently labeled

4E6G7 (Figure 8 J-L).

To ascertain the contribution of Fc

receptors, slices from adult (15-18 months)

htau/PS1 animals (n=3) were incubated with 0.5

μg/ml 4E6G7 for one hour at 37oC and increasing

concentrations of FcγII/III Receptor Block anti

CD16/CD32 monoclonal antibody (FC Block).

Relative to control, incubation with 0.05 and 0.5

μg/ml Fc Block resulted in a trend towards

reduced uptake (28% and 36% decrease) (p = 0.11

and 0.06 respectively). Significantly reduced

uptake was achieved with 5 and 50 μg/ml

concentrations of Fc Block with a 41 and 43%

reduction in final radioactivity (p = 0.05, and 0.04,

Figure 9A). Incubation with FcγRI (CD64)

antibody however, did not produce a significant


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change in final radioactivity at any of the

concentrations used. There was a trend towards

reduced uptake at the 50 μg/ml dose (p = 0.1)

indicating that there may be some minor

involvement of microglial uptake.

As with the slices, a significant decrease

in 4E6G7 uptake was observed in primary JNPL3

neurons at the 0.5, 5, and 10 μg/ml doses of Fc

Block (19%, 43%, and 53% decreases

respectively, p = 0.01, 0.006, and 0.008) (Figure

9B), whereas anti-FcγRI (0.05 – 10 μg/ml) did not

affect uptake at any dose (Figure 9B).

Incubation with 4E6G7 reduces intracellular

tau and this effect can be blocked with

dansylcadaverine

To determine if antibody entry into

neurons is a prerequisite for tau clearance, primary

neurons cultured from P0 JNPL3 pups were

incubated with 1, 10 or 20 μg/ml 4E6G7 with or

without 1 μg/ml dansylcadaverine to block

antibody uptake. The presence of intracellular

antibodies was visible by immunoblot using HRP-

conjugated mouse secondary antibody alone.

Uptake was linear up to 20 μg/ml for samples

incubated for 24 hours (data not shown). In

contrast, at 72 hours the difference between values

observed for 10 and 20 μg/ml was reduced

indicating that uptake may be saturating (Figure

10A). Similar to findings with radiolabeled

antibody, incubation with 1 µg/ml

dansylcadaverine significantly reduced uptake by

24% (p = 0.008; Figure 10B). To assess tau

clearance, immunoblots for total tau and Ser199

phosphorylated tau were performed. None of the

treatments significantly reduced actin levels. In

addition, an LDH assay was performed on media

taken from cultured cells treated with 4E6G7

alone or 4E6G7 and 1 μg/ml dansylcadaverine for

up to 7 days; again with no significant changes.

These data suggest that at short time points

antibody related cell death is minimal. No change

in tau signal was observed in any of the treatment

groups at 24 hours (data not shown). However, at

72 hours, 4E6G7 decreased total tau/actin ratio

and this effect was blocked by dansylcadaverine

(two way ANOVA – dose: p < 0.0001, treatment:

p < 0.0001, interaction: p < 0.0001). Post hoc

analysis revealed that all doses of 4E6G7 (1, 10

and 20 µg/ml) reduced total tau/actin (49, 61, and

58% respectively, p < 0.001) (Figure 11 A, C). In

contrast, samples incubated with both antibody

and dansylcadaverine showed no change in total

tau/actin ratio (Figure 11 B, C). These data show

that neuronal uptake of 4E6G7 is a prerequisite for

tau clearance within these primary neurons.

To assess if phosphorylated tau was

preferentially targeted for clearance, additional

immunoblots were performed with a polyclonal

antibody recognizing tau phosphorylated at Ser199

.

Previously, we have shown that treatment with

antibodies can reduce levels of phospho-tau

including epitopes other than the one targeted (11).

A similar pattern emerged as for total tau, albeit

less pronounced (two way ANOVA – treatment:

p=0.03, interaction: p=0.03). Post hoc analysis

showed significant 42% decrease in pSer199

/actin

at the highest dose of 4E6G7 (p<0.05), and this

effect was blocked by dansylcadaverine (Figure

11D). Further immunoblotting was performed with

antibodies recognizing tau phosphorylated at

Ser396

, and tau phosphorylated at Ser404

. However,

signal was not detectable indicating that the

presence of 4E6G7 may be masking the epitope.

These data suggest that while incubation with

4E6G7 reduces tau levels, it is not specifically

targeting phosphorylated tau for clearance at least

in primary tauopathy neurons.

Discussion

Our results show that neuronal uptake of

an anti tau IgG antibody, 4E6G7, is required for

tau clearance. It is dose dependent, saturable,

significantly reduced by temperature reduction,

and excess unlabeled IgG or 4E6G7. Furthermore,

internalization strongly correlates with levels of

pathological tau species. Because effects of

temperature on endocytosis are well established

(29-31), our results indicated that uptake was

likely an energy dependent process. Treatment

with clathrin mediated endocytosis inhibitors,

chlorpromazine and dansylcadaverine,

significantly reduced uptake in slice cultures and

primary neurons. Inhibition of phagocytosis had

no effect, indicating limited microglial uptake. We

further clarified the receptor type involved by

determining that uptake was partially blocked by

co-incubation with an Fc receptor antibody

(CD16/CD32), corresponding to FcII/III, in brain

slices and primary neurons, but not with anti-FcI.


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These results are consistent with previous findings

indicating that FcγII/III receptor mediated

endocytosis occurs via clathrin-dependent pathway

(32-34). No treatment completely blocked uptake,

however. This may be due to the nature of the

slice cultures, sectioning brains causes disruption

of some cell membranes. Other mechanisms may

also be involved such as fluid phase endocytosis,

although our data indicates this represents a

minority of uptake. Similar results were seen in

endothelial cells transfected with neonatal Fc

receptors (FcRn), in which fluorescently labeled

Fc was taken up via FcRn, and found in

compartments containing receptors, not co-

localized with dextran (35).

Interestingly, there was higher degree of

uptake in WT tissue than expected. In our prior

study, antibodies were only detected in brains of

Tg tauopathy mice and not WT animals, when

injected into the carotid artery (10). However, this

is likely due to an intact BBB in WT mice

preventing antibodies from crossing into the brain,

which is removed in slice cultures. Additionally,

slices are left to recover at room temperature,

which has been shown to increase tau

phosphorylation in animals (36), and in our

experiments results in a 28% increase in PHF-1

signal by immunoblot. Thus, phospho-tau levels in

slice cultures are higher than in the brains of WT

animals. Further, 4E6G7 binds to non-

phosphorylated tau as well, at least on ELISA

plates.

Involvement of FcRs in antibody uptake

and protein clearance has been examined utilizing

A antibodies in AD models with conflicting

reports. Early reports demonstrated that A

plaques and activated microglia in AD patients

were immunoreactive for antibodies against Fc I,

II and III (37) with similar results also seen in

Parkinson’s disease (38,39). Deane et al (2005)

indicated that FcR knock out animals showed

minimal clearance following administration of A

antibodies relative to FcR positive animals, and

additional data suggests that FcR binding is

important for amyloid clearance (40,41). In

contrast, other researchers found that in APP

transgenic animals, knocking out FcR did not

affect antibody clearance of plaques relative to

animals expressing Fc. It could be argued that

microglial uptake may be important in the

experiments described in this paper. For example,

antibody uptake highly correlates with the degree

of tau lesions. Such pathology may result in

greater activation of microglia, which may

phagocytose the antibody. Microglial activation

may then be a factor in increased 4E6G7 uptake.

However blocking phagocytosis did not

significantly affect uptake in either model system

utilized, indicating minimal involvement of

microglia, which should be abundant in slices and

present to some extent in primary cultures. Indeed,

we observe much less microglial activation in

various tauopathy mouse models than in A

plaque models because most tau lesions are

intracellular. Additionally, in brain slice cultures

the majority of fluorescently labeled 4E6G7 was

found in neurons, with some uptake in microglia

but none in other glial cell types.1 Further, data

from primary neuronal cultures showed that

uptake is primarily occurring via receptor-

mediated clathrin dependent endocytosis and to a

large extent by FCII/III receptors.

In addition to expression on glial cells,

neurons also express Fcreceptors. Earlier

findings indicated the presence of FcI receptors

on sensory neurons, but not II or III (42).

However, more recent studies have found

expression of FcIIb on Purkinje cells and

parvalbumin neurons, and expression of I, II, III,

and IV in primary neuronal cultures (43-45).

Additionally, neurons are responsive to

extracellular IgG by upregulating FcR (43).

These results, and data showing that antibodies

against pathological proteins are taken up by

neurons, suggest a mechanism by which the FcR

contributes to protein clearance in immunotherapy

(10,19-22). Our results show that anti-tau antibody

uptake can be blocked to a large extent using

FcII/III antibodies suggesting that low affinity Fc

receptors are the major route into neurons.

Of particular note is the finding that, in

cultured neurons, partial blockage of receptor

mediated endocytosis with dansylcadaverine is

sufficient to prevent tau clearance. Importantly,

this indicates that antibody-mediated tau clearance

takes place primarily within neurons. Previous

data from our lab showed that antibodies were

present in neurons of treated animals, and that the

treatment resulted in clearance of phospho-tau but

not total tau (10), which we subsequently


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confirmed in other studies (11,12,19). Together,

these prior studies suggest involvement of

intracellular tau antibodies in tau clearance, and

we now show in the primary Tg JNPL3 neuronal

model that this pathway is necessary for removal

of tau. The main difference between those prior

animal studies and this experiment is an observed

tau antibody mediated decrease in total tau in

culture and not in animals. This discrepancy may

be explained by numerous differences in these

models and study design, including antibodies and

their concentration per tissue weight which is

likely much greater in the culture. Our findings

that blocking tau antibody uptake prevented tau

clearance indicate that the intracellular clearance

pathway is the primary one, at least in this model

and under relatively acute conditions.

Additionally, this treatment does not appear toxic

at the concentrations used. LDH assays revealed

no change in neurons treated with 4E6G7 alone or

4E6G7 and dansylcadaverine relative to control

cells at up to 7 days incubation. Actin levels were

also not significantly altered at any of the

conditions used.

The involvement of FcR, coupled with our

data showing uptake-dependent intracellular tau

clearance, also suggests a pathway for improving

efficacy of immunotherapy. In Aβ-targeting

experiments, antibodies with isotypes having

higher affinity for phagocytic FcRs led to better

clearance (40). Indeed, affinity for Fc receptors

was more important for clearance than affinity for

A. This suggests selection of antibodies could be

improved by focusing on specific isotypes or

modifications that would improve receptor

binding. For example, mutations in antibody Fc

region are capable of increasing affinity for

neonatal FcRs (46), whereas other mutations can

shift the ratio of FcRIIa/FcRIIb affinity leading

activation of macrophages (47). Thus it is possible

that antibodies containing mutations that enhance

binding to the desired receptor may provide

greater efficacy in clearing protein aggregates. It is

uncertain how or if such mutations would affect

neuronal uptake, but further studies are clearly

warranted.

Besides intracellular aggregates,

extracellular tau may play an important role in

disease progression. Recent findings have

demonstrated that tau pathology can spread across

synapses and that pathological tau can be taken up

by cells (17,48-57). Cultured cells, even healthy

ones, have also been found to secrete

phosphorylated tau (48), and extracellular tau

increases intracellular calcium in cultured neurons

(58). This presents an additional mechanism by

which treatment with antibodies might alter

disease progression. Antibodies may prevent

secretion by targeting intracellular tau into protein

degradation pathways. Extracellularly, antibodies

may bind to tau and promote its uptake into

microglia or neurons for clearance and thereby

prevent spread of tau pathology to neighboring

neurons.

Previously our lab has shown that tau

antibodies clear tau aggregates and improve

cognition and other tau-related deficits in

tauopathy mouse models. Also, these antibodies

not only cross the blood brain barrier but enter

neurons. Herein, we have identified the

mechanism of this neuronal uptake showing it to

be primarily receptor-mediated via FcII/III

receptors, to correlate very well with the degree of

tau pathology, and to be a prerequisite for tau

clearance. These important findings identify a

neuronal pathway which may be activated in

response to intraneuronal tau aggregation, and that

can be manipulated to improve the efficacy of

immunotherapy.


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Footnotes

1 Unpublished observation presented by Gu and Sigurdsson at Society of Neuroscience Annual Meeting

2012, poster number 852.12/E24: Efficacy and mechanisms of antibody-mediated clearance of tau

aggregates in a brain slice model.

Acknowledgements

This work was funded with support from NIH grant numbers NS077239, AG032611 and AG020197.

NYU technology on tau immunotherapy is licensed to and is being co-developed with H. Lundbeck A/S.

Drs. Frank LaFerla and Salvatore Oddo provided the breeding pair of 3xTg mice, and Dr. Peter Davies

supplied the PHF-1 antibody. Dr. Allal Boutajangout was responsible for initiating the htau/PS1 mouse

line.


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Figure Legends

Figure 1. 4E6G7 stains pathological tau in sections from transgenic animals

A. Brains from 3xTg mice (aged 22 months) were sectioned and incubated with a 1:2000 dilution of

purified 4E6G7. Biotinylated mouse secondary and DAB were used to visualize the antibody.

Represenatative images from coronal sections of the subiculum show extensive staining of pathological

tau in the neuronal cell bodies. B. Adjacent sections from the same animal stained with PHF-1 show

neuronal tau aggregates as well. The pattern of staining with PHF-1 is less punctate than that obtained

with 4E6G7. C. An age matched wild type animal from the same line was also incubated with 4E6G7 and

showed no reactivity. All images were collected at 100X magnification.

Figure 2. 4E6G7 uptake is rapid and saturable

Brain sections prepared from htau/PS1 mice (n = 3, 15 – 17 months) were incubated with increasing


-4E6G7 antibody (Ab). Concentrations of antibody in buffer ranged from 0.01 g to

5 g per ml of buffer. A. Sections were removed and assayed for radioactivity at 30, 60 and 120 minutes.

Similar values were obtained for each time point indicating that uptake is rapid, occurring within 30

minutes of incubation. All points represent an average ± SEM. B. As above, brain slices from htau/PS1

animals (15 – 17 months) were incubated with increasing concentrations of I125

-4E6G7. In this

experiment, an additional 10 g/ml dose was added and all samples were collected after 60 minutes of

incubation. Uptake of the antibody appears linear to 5 g/ml, but saturates at higher concentrations. C.

Similar results are obtained utilizing fluorescently labeled 4E6G7 with slice cultures prepared from

JNPL3 mice (n = 3, 3-4 months). D. Slices from wild type animals (n = 4, 12-17 months) were incubated

with I125

-4E6G7 at increasing concentrations for 60 minutes. Similar to both transgenic lines, uptake

saturated between 5 and 10 g/ml.

Figure 3. Slice cultures are enriched in phospho-tau relative to flash frozen tissue.

Tissue from wild type animals (n = 3) was collected to assess the effect of slice culture preparation

method on the levels of phosphorylated tau. Control hemispheres (C) were prepared as normal. The other

hemisphere was flash frozen on dry ice (F). A. Immunoblots of both hemispheres were probed with

antibodies against total tau (Dako, rabbit polyclonal) and PHF-1. B. Flash freezing did not significantly

change total tau levels relative to control hemispheres. C. PHF-1 signal was reduced in flash frozen

hemispheres by 26% (p = 0.006). D. Controlling for total tau levels, chemiluminescent signal in flash

frozen tissue was 28% lower than for slices allowed to incubate at room temperature (p = 0.01).

All bars represent averages ± SEM ** p ≤ 0.01

Figure 4. Excess unlabeled IgG or 4E6G7 reduces uptake of labeled 4E6G7

A. Slices were prepared as described from htau/PS1 mice. Brain sections were separated into three

groups: control (0.5 g/ml I125

-4E6G7 alone) or 0.5 µg/ml 4E6G7 plus excess unlabeled mouse IgG1

(either 5 or 50 g/ml). Averages and SEM were determined for each treatment group. Addition of 10x

excess IgG to the culture media significantly reduced the levels of radioactivity in the tissue (41%

decrease, p = 0.04). Incubation with 100x IgG also produced a significant decrease relative to control

(40% decrease, p = 0.02), but was not significantly reduced relative to 10x treated slices. B. Similar

results were obtained using sections from JNPL3 mice and fluorescently labeled 4E6G7 (29% decrease, p

= 0.05). C. Slices were prepared as described and incubated with 0.05 g/ml I125

-4E6G7, and either 5 or

50 g/ml unlabeled 4E6G7. Significant reductions were seen in final radioactivity at both doses (35% and

61%, p = 0.01 and 0.005 respectively). Unlike IgG samples, 50 g/ml of unlabeled antibody significantly

reduced radioactivity relative to the 5 g/ml concentration (p = 0.001).


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Bars represent average ± SEM * p ≤ 0.05, ** p ≤ 0.01

Figure 5. 4E6G7 uptake correlates with pathological tau levels

A, B. Immunoblots using a pan tau antibody (Dako) and PHF-1 were carried out on the total and sarkosyl

insoluble (aggregated) fractions respectively from three different mouse lines (n = 3 mice per group). C.

Total tau levels from the low speed supernatant were quantified for each of the different mouse lines used.

Bars represent average ± SEM. D. The sarkosyl insoluble fraction was isolated, and chemiluminescence

was determined. Values were corrected for background and the average values obtained. Bars represent

average ± SEM E. Slices prepared from the same htau/PS1, 3xTg and WT mice were incubated for one

hour in varying concentrations of I125

-4E6G7 (n = 3 mice per group). Average radioactivity per mg of

tissue was determined as described above, and values were fit to a linear curve (squares htau/PS1, circles

3xTg, triangles WT). All points represent averages ± SEM. F. Chemiluminescent signal was plotted

against radioactivity for each sample (total n = 9). A linear regression was fit to the data yielding r2 of

0.77 (p = 0.02). G. Chemiluminescent signal from the sarkosyl insoluble fraction was plotted against final

radioactivity for each animal (total n = 9) and fit to a linear regression. An r2 of 0.87 (p = 0.002) was

obtained indicating that levels of pathological tau and antibody uptake are highly correlated. H.

Additional sections from htau/PS1 and WT mice were incubated with I125

-IgG1Samples were

processed as described. IgG uptake was greater in slice cultures made from htau/PS1 relative to WT, but

both were substantially less than 4E6G7 uptake.

Figure 6. Specific inhibitors of clathrin mediated endocytosis (CME) reduce 4E6G7 uptake

A. Sections from htau/PS1 mice (n = 3 per group) were incubated for 1 hour with 0.5 g/ml I125

-4E6G7

and increasing concentrations of endocytosis inhibitors. No change in uptake was observed with filipin III

(caveolin specific). Incubation with chlorpromazine (clathrin specific) significantly reduced final

radioactivity at high concentrations (10, 50, 100 g/ml) (15-34% reduction, p = 0.03). Phagocytosis

inhibitor cytochalasin D did not alter uptake. B. Primary neurons (n = 6 wells per group) were cultured

from JNPL3 mice and incubated with I125

-4E6G7 and increasing concentrations (0.5 – 50 g/ml) of

endocytosis inhibitors. Clathrin specific inhibitor dansylcadaverine significantly inhibited uptake at every

dose (36-78% reduction, p = 0.00006-0.000005). Filipin III produced a 30% reduction (p = 0.04) at the

highest dose, the only one to significantly alter uptake. Cytochalasin D did not affect radioactivity at any

dose. All bars and markers represent average ± SEM. * p ≤ 0.05, *** p ≤ 0.001

Figure 7. Fluid phase endocytosis is not the major route of uptake

A,B,C. Primary neuronal cultures from postnatal day 0 JNPL3 mice were incubated for one hour with

both fluorscently labeled dextran (488 nm) and 4E6G7 (568 nm). Coverslips were fixed and mounted, and

images collected using a Nikon Eclipse Ti confocal microscope. D. The number of puncta positive for

dextran, 4E6G7, or both were counted (~1700 puncta). Only 17 ± 1.2 % of all the puncta were positive

for both, of those positive for 4E6G7 (red or yellow) 27 ± 1.4 % were co-localized.

Figure 8. Primary neuronal cultures and brain slices take up 4E6G7 and stain positive for Fc II/III

receptors. A. Representative images of cells positive for Fcγ II/III receptors. Receptors are found

throughout the cells. B. Primary neurons stained with Dako rabbit polyclonal pan-tau antibody showing

strong staining in neuronal cultures. C. Merged image. D. Representative images of sections from JNPL3

mice stained for Fcγ II/III showing similar results to those seen in neuronal cultures. E. Pan-tau antibody

staining showing cell body and neuropil staining. Tau aggregates are also visible on the inner face of the

plasma membrane in neurons. F. Merged image. G. Fluorescently labeled 4E6G7 uptake in primary

neurons. 4E6G7 is seen as bright puncta within the neuron. H. Pan-tau antibody showing strong staining

in cultured neurons similar to panel B. I. Merged image. J. Fluorescently labeled 4E6G7 uptake in brain

slice cultures showing a similar pattern of 4E6G7 distribution to that in G. K. Pan tau antibody staining in

brain slices showing results similar to panel E. L. Merged image.


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Figure 9. Blocking Fc II/III receptors reduces uptake

A. Slices from htau/PS1 (n = 3, 15-18 months) were incubated with 0.5 g/ml I125

-4E6G7 and increasing

concentrations of Fc Block (anti CD16/CD32, FcγII/III). Fc Block at 0.05 and 0.5 g/ml produced a trend

towards reduced radioactivity. Both 5 and 50 g/ml concentrations of Fc Block resulted in significantly

reduced uptake (41% and 43% reduction, p = 0.05, and 0.04 respectively). The same protocol was used

with an Fcγ I antibody (anti-CD64) with no significant reduction in final radioactivity. B. Primary

neurons (n = 6 wells per group) from JNPL3 pups were also incubated with Fc Block and I125

-4E6G7. The

1, 5 and 10 g/ml antibody concentrations significantly reduced uptake (19%, 43%, and 53%

respectively, p = 0.01, 0.006, and 0.008). As with slice cultures, incubation with Fc I antibodies did not

significantly affect uptake at any dose.

Bars and markers represent average ± SEM. * p ≤ 0.05, ** p ≤ 0.01

Figure 10. 4E6G7 uptake into cells can be visualized by immunoblot and is inhibited by

dansylcadaverine

Primary JNPL3 neurons (n = 6 wells per group) were incubated with 4E6G7 at three different

concentrations 1, 10, or 20 g/ml. Samples were collected and processed for immunoblotting as

described. Blots were incubated with mouse secondary alone and chemiluminescence signal determined

for each. A. Antibody uptake is dose dependent with antibody accumulating in the cells with increased

dose. B. Samples incubated with 10 g/ml 4E6G7 in the presence or absence of dansylcadaverine (DC)

were compared to assess the effects of blocking clathrin mediated endocytosis on antibody uptake.

Uptake was reduced by 24% with DC present (p = 0.008; all samples run on the same gel). Bars represent

average ± SEM. ** p < 0.01

Figure 11. 4E6G7 reduces intracellular tau and blocking antibody uptake eliminates its

effectiveness

Primary JNPL3 neurons (n = 6 wells per group) were incubated with 1, 10, or 20 g/ml 4E6G7 for 72

hours in the presence or absence of 1 g/ml dansylcadaverine (DC). A,B. Immunoblots against total tau,

pSer199, and actin were carried out and two way ANOVAs performed. C. Analysis with two way

ANOVA revealed significant effects of dose, presence of DC, as well as interaction effects (p< 0.0001 for

all). 4E6G7 alone produced significant reductions in intracellular tau levels relative to actin levels at all of

the doses used (49, 61, and 58 % respectively, p < 0.001). Addition of 1 g/ml DC blocked tau reduction.

D. Two way ANOVA testing of Ser199

data revealed significant effects of DC and interaction effects (p =

0.03). Phospho-tau levels were significantly reduced at 20 µg/ml 4E6G7 (p < 0.05). All points represent

average ± SEM . * p ≤ 0.05, *** p ≤ 0.001


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Figure 1


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Figure 9


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Figure 10


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Figure 11


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Erin E. Congdon, Jiaping Gu, Hameetha B. R. Sait and Einar M. SigurdssonReceptor Endocytosis, and is a Prerequisite for Acute Tau Clearance

γAntibody Uptake into Neurons Occurs Primarily via Clathrin Dependent Fc

published online October 25, 2013J. Biol. Chem.

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neuronal antibody uptake and tau clearance

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