Intramuscular injection of vectorized-scFvMC1 reduces pathological tau in two different tau transgenic models

Abstract

Abstract With evidence supporting the prion-like spreading of extracellular tau as a mechanism for the initiation and progression of Alzheimer’s disease (AD), immunotherapy has emerged as a potential disease-modifying strategy to target tau. Many studies have proven effective to clear pathological tau species in animal models of AD, and several clinical trials using conventional immunotherapy with anti-tau native antibodies are currently active. We have previously generated a vectorized scFv derived from the conformation-dependent anti-tau antibody MC1, scFvMC1, and demonstrated that its intracranial injection was able to prevent tau pathology in adult tau mice. Here, we show that, in a prevention paradigm and in two different tau transgenic models (JNPL3 and P301S), a one-time intramuscular injection of AAV1-scFvMC1 generated a long-lasting peripheral source of anti-tau scFvMC1 and significantly reduced insoluble and soluble tau species in the brain. Moreover, our data showed that scFvMC1 was internalized by the microglia, in the absence of overt inflammation. This study demonstrates the efficacy of intramuscular delivery of vectorized scFv to target tau, and suggests a new potential application to treat AD and the other tauopathies.

Full text

R E S E A R C H Open Access
Intramuscular injection of vectorized-
scFvMC1 reduces pathological tau in two
different tau transgenic models
Francesca Vitale
1
, Jasmin Ortolan
1
, Bruce T. Volpe
2,3
, Philippe Marambaud
1,2
, Luca Giliberto
1,2,4*
and
Cristina d’Abramo
1,2*
Abstract
With evidence supporting the prion-like spreading of extracellular tau as a mechanism for the initiation and
progression of Alzheimer’s disease (AD), immunotherapy has emerged as a potential disease-modifying strategy to
target tau. Many studies have proven effective to clear pathological tau species in animal models of AD, and several
clinical trials using conventional immunotherapy with anti-tau native antibodies are currently active. We have
previously generated a vectorized scFv derived from the conformation-dependent anti-tau antibody MC1, scFvMC1,
and demonstrated that its intracranial injection was able to prevent tau pathology in adult tau mice. Here, we show
that, in a prevention paradigm and in two different tau transgenic models (JNPL3 and P301S), a one-time
intramuscular injection of AAV1-scFvMC1 generated a long-lasting peripheral source of anti-tau scFvMC1 and
significantly reduced insoluble and soluble tau species in the brain. Moreover, our data showed that scFvMC1 was
internalized by the microglia, in the absence of overt inflammation. This study demonstrates the efficacy of
intramuscular delivery of vectorized scFv to target tau, and suggests a new potential application to treat AD and
the other tauopathies.
Keywords: Vectorized antibodies, AAV, scFv, Tau, Immunotherapy, Intramuscular injection
Introduction
The microtubule-associated protein tau plays a physio-
logical role in microtubule stabilization, axonal growth
and cytoskeletal dynamics in neurons, but its aggrega-
tion characterizes several neurological diseases classified
as tauopathies, including Alzheimer’s disease (AD) [1–
5]. As increasing evidence supports the existence of tau
as an extracellular protein and the concept of its trans-
cellular propagation as a mechanism for the initiation
and progression of AD, tau has become an attractive tar-
get for immunotherapy in animal models of AD and
related tauopathies [6–13]. A conspicuous amount of
data has been produced by different laboratories, includ-
ing ours, showing reduction of tau pathology in trans-
genic animal models using tau monoclonal antibodies,
with a different degree of success according to which
epitopes were targeted [14–26]. However, passive im-
munotherapy using conventional antibodies in humans
presents several potential limitations such as low perme-
ability to cross the blood-brain barrier (BBB), detrimen-
tal inflammatory reactions and microhemorrhages
associated to the treatment, requirement for repeated
dosing, patients compliance and high costs [27–30]. To
overcome these issues, several groups have engineered
antibodies as fragments, i.e. single chain variable frag-
ment (scFv), to be used in combination with gene deliv-
ery strategies based on the use of viral vectors:
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data made available in this article, unless otherwise stated in a credit line to the data.
* Correspondence: lgiliberto@northwell.edu;cdabramo@northwell.edu
1
Institute of Molecular Medicine, The Litwin-Zucker Center for Alzheimer’s
Disease & Memory Disorder, The Feintein Institutes for Medical Research,
Manhasset, NY, USA
Full list of author information is available at the end of the article
Vitale et al. Acta Neuropathologica Communications (2020) 8:126
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vectorized anti-Aß (amyloid-beta) and anti-tau scFvs
have previously shown benefits in models of AD [31–
45]. ScFvs consist of the smallest functional antigen-
binding domain of an antibody (Ab) exhibiting compar-
able antigen-binding affinity as the parent immuno-
globulin, reduced size, improved pharmacokinetic in
terms of tissue penetration and lack of an Fc receptor-
mediated inflammatory response [46–48]. Due to their
short systemic half-life in vivo [49,50], in order to reach
a sustained and long-lasting expression scFv are gener-
ally cloned in adeno associated viral vectors (AAVs) and
delivered by one-time injections [34,46,47]. We have
previously shown [43] that the AAV vectorized-
scFvMC1, a recombinant version of the native anti-tau
conformational mAb (monoclonal antibody) MC1, sig-
nificantly reduces brain pathological tau in adult JNPL3
mice, by one-time intracranial injection. In parallel with
AAV-based delivery, Spencer et al. [44] have shown that
systemic injection of a lentiviral vector (LV) carrying a
scFv directed to 3Rtau and enhanced for brain penetra-
tion (LV-3RT-apoB) was able to reduce tau accumula-
tion, neurodegeneration and behavioral deficit in a tau
transgenic model. Although using third-generation lenti-
viral vectors has emerged as a promising therapeutic op-
tion for conditions as primary immunodeficiencies and
cancers, it is still necessary to understand the long-term
safety and efficacy of these vectors in humans, especially
in respect to their potential for insertional oncogenesis
[51]. In this context, using AAVs is considered safer
than LVs.
Direct delivery of AAV into the brain has been tested
in a number of clinical trials [52–57] and parallel efforts
have been done over the past years to develop new
brain-targeted AAVs to treat CNS diseases in vivo using
a systemic delivery approach [58]. In this respect, per-
ipheral administration provides obvious advantages to
AAV gene therapy: a non-invasive route of injection,
lack of surgery-related side effects, improved patient
compliance and costs. In this study we propose to de-
velop a novel therapeutic approach for AD and tauopa-
thies by using intramuscular (IM) delivery of AAV-
vectorized scFv. To date, there has been little effort to
develop peripheral protocols targeting skeletal muscle to
tackle CNS disorders. IM injection provides a quick,
easy, non-invasive and safe route of administration, and
can routinely be performed in virtually any setting. Also,
skeletal muscles are an ideal target tissue for AAV trans-
duction because individual fibers are large, multinucle-
ated and with minimal cellular turnover [59]. Taking
advantage of the practical features and biological proper-
ties of this delivery approach, the aim of the present
study was to generate a long-lasting peripheral niche
able to produce and release anti-tau scFv in the circula-
tion to target cerebral tau, without transducing vital
organs such as liver, kidneys and heart. A similar strat-
egy has been previously tested in a mouse model of AD,
where intramuscular delivery of AAV1 vectored anti-Aß
scFv was able to reduce Aß load in brain [35,45]. Simi-
larly, gene therapy for Alpha-1 antitrypsin (AAT) defi-
ciency has been developed in humans using
recombinant AAV1 serotype, demonstrating a continued
stable transgene expression at 5 years after transduction
[60,61].
In this study, for the first time, we demonstrate the
in vivo feasibility and efficacy of targeting pathological
tau in the brain, by employing intramuscular delivery of
vectorized anti-tau scFv. Two different tau transgenic
models, the JNPL3 and P301S mice, received a single IM
injection of AAV1-scFvMC1, showing a significant re-
duction of tau pathology, with some differences between
strains. Moreover, no signs of inflammation were ob-
served upon AAV1-scFvMC1 immunization, showing
that tau clearance does not involve Fc-receptor-
mediated and microglia-associated detrimental inflam-
matory response. However, our in vitro and in vivo data
point to the microglia as a player in the uptake and
clearance of scFv-tau, despite the absence of Fc, adding
to the potential mechanisms of action of tau
immunotherapy.
In summary, our data support the peripheral intramus-
cular route as an effective, feasible and safe delivery ap-
proach for AAV-scFv-based anti tau immunotherapy,
with relevant translational potential applications to other
tauopathies and brain disorders.
Methods
ScFv-MC1 design and sub-cloning into AAV1
The light and heavy-chain variable domains correspond-
ing to the MC1 antibody were sequenced employing the
MCLAB antibody service (San Francisco, CA). As previ-
ously published [43] the V
H
and V
L
chains were joined
together by a 15 amino acid residues linker (Gly
4
Ser)
3
.
5′-terminal signal peptide (SP) and 3′-terminal Myc and
His6X tags were added. The AAV packaging and purifi-
cation service was provided by Vector Biolab (Malvern,
PA). ScFv-MC1 was sub-cloned into the adeno-
associated viral vector serotype 1 (AAV1) under the con-
trol of the synthetic strong CAG (CMV-chicken beta
actin-rabbit beta globin) promoter. In order to enhance
expression of the transgene, the WPRE Woodchuck
hepatitis virus (WPRE) post-transcriptional regulatory
element was added 5′of the Myc and His6X tags.
Tau transgenic mice
JNPL3 mice obtained from Taconic (Germantown, NY)
express 0N4R human tau with the P301L mutation that
causes frontotemporal dementia in humans, under the
mouse prion promoter. JNPL3 mice develop NFTs-like
Vitale et al. Acta Neuropathologica Communications (2020) 8:126 Page 2 of 19
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pathology as early as 4.5 months and in later stages pro-
gressive deterioration of the motor function [62]. Homo-
zygous P301S were obtained from Dr. Michel Goedert
(Cambridge, UK) [63]: these mice, on pure C57BL/6
background, express 0N4R human tau carrying the
P301S mutation, under the control of the neuron-
specific murine Thy-1 promoter, and they develop wide-
spread tau pathology affecting cerebral cortex, hippo-
campus and brain stem as early as 6 months, and partial
paralysis of the lower limbs by 8 months of age. Animals
were treated according to the current regulations for the
proper handling of research animals, following an ap-
proved IACUC protocol.
ScFvMC1 purification
ScFvMC1 purification was performed as previously pub-
lished [43]. Briefly, scFv-MC1 was cloned into the mam-
malian expression vector pcDNA3.1 (Genewiz, South
Plainfield, NJ) and transfected into HEK293T, using Li-
pofectamine 2000 (Invitrogen, Carlsbad, CA). After 48 h
of transfection, the scFv released into the conditioned
medium was affinity purified using a Ni-Sepharose High
Performance column (GE Healthcare, Port Washington,
NY). The efficiency of purification was tested using an
immunosorbent assay employed to assess the antigen–
binding specificity of the scFvMC1, as previously de-
scribed [43]. Starting material, flow through and eluted
fractions were tested to check for proper enrichment of
the purified material. The purified scFv-MC1 was
checked on Coomassie-stained SDS-PAGE gel for proper
molecular weight.
Infrared conjugation and intravenous (IV) injections
ScFv-MC1 and MC1 have been conjugated with IRDye
800CW, using IRDye 800CW protein Labeling Kit Low-
MW or High-MW respectively (LI-COR Biosciences,
Lincoln, NE), according to the manufacturer instruc-
tions. Briefly, scFv-MC1 and MC1 were dialyzed in 50
mM potassium phosphate buffer pH 8.5 at 4 °C, over-
night; the pH was then adjusted with 1 M potassium
phosphate buffer to 9. After 2 h incubation, the unconju-
gated dye was removed using desalting spin columns
(Zeba Desalt Spin Columns, Thermo Scientific).
ScFvMC1 and MC1 antigen-binding reactivities were
measured against an MC1 specific peptide [43] by im-
munosorbent assay, in order to exclude loss of activity
upon conjugation.
To verify the ability of scFvMC1 and MC1 to cross the
blood brain barrier, IV injection was performed in 3-
month-old JNPL3 mice using 100 μg of the antibodies:
saline, scFvMC1-IRDye, MC1-IRDye, or unlabeled anti-
bodies were injected (n= 3 per group). Mice were anes-
thetized with isoflurane and the injections performed
retro-orbitally. Mice were sacrificed 2 h post injection;
brains were harvested and dissected into cortex (Ctx),
hindbrain (HB) and hippocampus (Hip).
Homogenization was performed in 1X RIPA buffer
(Thermo Fisher Scientific, Waltham, MA) with the Mini
protease inhibitor cocktail (Roche, Indianapolis, IN).
Brain samples from each region were spotted on
0.45 μm nitrocellulose followed by IR signal acquisition
at 789 nm, using Sapphire Biomolecular Imager (Azure
Biosystems, Dublin, CA).
Intra-muscular (IM) injections
AAV1-CAG-scFvMC1 or AAV1-CAG-eGFP were
injected at a dose of 2X10
11
GC per mouse. Each AAV
was diluted in PBS at a final volume of 50 μl, and a one-
time intramuscular injection was administrated in the
right tibialis. Injections were performed upon anesthesia
with isoflurane.
Twenty-six females JNPL3 (n = 13 per group) mice
were injected at 3 month of age and sacrificed 4 months
later. The P301S line was injected at 2 month of age and
sacrificed 4 months later; we used twelve females P301S
in total (n= 6 per group). Overall, 26 JNPL3 and 12
P301S mice were employed in this study.
Brain extracts and tissues preparation
Mice were sacrificed by isoflurane overdose, decapitated
and processed as described previously [14]. The brain
was removed and divided at the midline so that just one
half of the brain was dissected for biochemical analysis.
Cortex, hippocampus and hindbrain were homogenized
separately using an appropriate volume of homogenizing
buffer, a solution of Tris-buffered saline (TBS), pH 7.4,
containing 10 mM NaF, 1 mM Na
3
VO
4
and 2 mM
EGTA, plus the complete Mini protease inhibitor cock-
tail (Roche). Supernatants were analyzed for protein con-
centration using DC Protein Assay (Bio-Rad
Laboratories, Hercules, CA). Brain homogenates were
stored at −80 °C and used for separate measurement of
soluble and insoluble tau. Soluble tau was measured as
heat-stable preparation (hsp) from brain. Hsp were pre-
pared by adding 5% ß-Mercaptoethanol and 200 mM
NaCl to the brain homogenates. Samples were then
heated at 100 °C for 10 min and cooled at 4 °C for 30
min. After centrifuging at 14,000 g in a table-top micro-
centrifuge at 4 °C for 15 min, supernatants were col-
lected and 5X sample buffer (Tris-buffered saline, pH 6.8
containing 4% SDS, 2% ß-Mercaptoethanol, 40% glycerol
and 0.1% bromophenol blue) was added. To obtain in-
soluble tau preparations (INS), homogenates were
thawed and spun at 14,000 g for 10 min at 4 °C. The col-
lected supernatants were centrifuged at 200,000 g for 30
min at 4 °C; the pellets were then re-suspended in hom-
ogenizing buffer and centrifuged again at 200,000 g for
30 min at 4 °C. The final pellets were re-suspended in 1X
Vitale et al. Acta Neuropathologica Communications (2020) 8:126 Page 3 of 19
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sample buffer and heated at 100 °C for 10 min to effi-
ciently dissociate the insoluble tau fraction.
Liver, kidney and heart were harvest and homogenated
using 1X RIPA buffer with the complete Mini protease
inhibitor cocktail (Roche). Protein concentration were
analyzed using DC Protein Assay (Bio-Rad Laboratories)
and samples were prepared for western blotting. For tibi-
alis and gluteus maximum muscles protein were ex-
tracted in skeletal muscle homogenizing buffer (20 mM
Tris, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1% Tri-
ton X-100, 10% glycerol, 1 mM EDTA and 1 mM dithio-
threitol) plus the complete Mini protease inhibitor
cocktail (Roche). Tissue were mince using a Dounceho-
mogenizer, sonicated and then let vortexed overnight at
4 °C. The supernatant, containing the protein extract,
was collected after 15 min centrifugation at 14,000 g and
used in immunoblot, as described later.
Tau ELISA
Levels of total and phosphorylated tau were assessed
using the Low-tau ELISA (enzyme-linked immunosorb-
ent assay) protocol previously published [64,65]. 96-well
plates were coated for 48 h at 4 °C with specific purified
monoclonal tau antibodies (DA31, CP13, PHF1, RZ3) at
a concentration of 6 μg/ml. After washing, plates were
blocked for 1 h at RT using StartingBlock buffer
(Thermo Fisher Scientific). Brain samples and standards
were diluted in 20% SuperBlock buffer (Thermo Fisher
Scientific) in 1XTBS and loaded on the plates. Once the
samples were added, the total tau detection antibody
DA9-HRP, diluted 1:50 in 20% SuperBlock in 1XTBS,
was added to the samples and tapped to combine. Plates
were then incubated overnight at 4 °C. Next day, 1-Step
ULTRA TMB-ELISA (Thermo Fisher Scientific) was
added for 30 min at RT, followed by 2 N H
2
SO
4
to stop
the reaction. Plates were read with Infinite m200 plate
reader (Tecan, San Jose, CA) at 450 nm.
Immunoblotting
An aliquot of the total lysates was used for western blot-
ting (WB). 0.1% SDS was added to the lysates, followed
by sonication (3 cycles, 10 s each). Samples were run on
4–20% Criterion Tris-HCl gels (Bio-Rad Laboratories)
and electrophoretically transferred to a nitrocellulose
membrane (Thermo Fisher Scientific). Residual protein-
binding sites were blocked by incubation with 5% non-
fat milk in 1XTBST (1X TBS plus 0.1% Tween 20) 1 h at
RT, followed by an overnight (O/N) incubation at 4 °C
with primary antibodies diluted in 20% SuperBlock buf-
fer (Thermo Fisher Scientific) in 1XTBST. Mouse anti-
tubulin (Thermo Fisher Scientific) were diluted 1:5000;
anti-Myc-tag 9B11 (Cell Signaling, Danvers, MA) was di-
luted 1:1000. Appropriate isotypes secondary antibodies
HRP-conjugated were diluted 1:2000 or 1:10000 in 5%
non-fat milk 1XTBST, and added for 1 h at RT. Every
step was followed by 3 or 4 washes in 1X TBST. Detec-
tion was performed using Pierce ECL Western Blotting
Substrate (Thermo Fisher Scientific) or SuperSignal
West Dura extended duration substrate (Thermo Fisher
Scientific) and exposed to x-ray films.
Immunocytochemistry, immunofluorescence and image
analysis
Tau staining and immunofluorescence were performed
according to standardized protocols [14,43]. After de-
capitation, half of the brain was fixed overnight in 4%
paraformaldehyde at 4 °C. Serial sections were cut from
the fixed brain half on a vibratome, conserved in TBS
(50 mM Tris, 150 mM NaCl, pH 7.6)/0.01% NaN
3
, and
stained on 24-well plates with a panel of tau antibodies.
Endogenous peroxidases were quenched with 3% H
2
O
2
/
0.25% Triton X-100/1XTBS for 30 min. Non-specific
binding was blocked with 5% non-fat milk-1XTBS for 1
h at RT. Primary antibodies were used as follows: anti
tau antibodies RZ3 and MC1 (1:500), CP13 and PHF1
(1:5000); all antibodies were diluted in 5% non-fat milk-
1XTBS, and incubated O/N at 4 °C, shaking. Biotin-
conjugated secondary antibodies (SouthernBiotech, Bir-
mingham, AL) directed against the specific isotypes were
diluted 1:1000 in 20% SuperBlock, left for 2 h at RT, and
lately Streptavidin-HRP (SouthernBiotech) was incu-
bated for 1 h. Staining was visualized by 3,3′-Diamino-
benzidine (Sigma-Aldrich, St. Louis, MO). Images were
acquired using Olympus BH-2 bright field microscope
(Waltham, MA); analyzed and processed using ImageJ/
Fiji software (NIH). Semi-quantification was done on the
hippocampal quadrant CA1 and on the entorhinal cortex
by using the measure particles tool, working with 8-bit
images and adjusting the threshold.
For immunofluorescence, sections were pre-incubated
5 min at RT in 1XTBS (Gibco, Carlsbad, CA) containing
0.2% TritonX100 (Sigma-Aldrich). After blocking 1 h at
RT with a solution containing 5% normal goat serum
(Sigma-Aldrich) diluted in 1XTBS/0.2% Triton, sections
were incubated with primary antibodies diluted in 1%
normal goat serum in 1XTBS/0.2% Triton: anti-Myc-
Alexa Flour555 1:500 (EMD Millipore), Iba-1 1:1000
(Wako Chemicals, Richmond, VA), anti-CD68 1:200
(Bio-Rad Laboratories) and RZ3 (anti-tau pThr231) 1:
500. After washing 3X in 1XTBS/0.2% TritonX100,
Alexa Fluor secondary antibodies −488 and −568 and −
350 (Invitrogen) were added at 1:1000 or 1:2000 dilu-
tions for 1 h at RT, in different combinations in order to
obtain multiple labeling images. DAPI (Invitrogen) was
used to counterstain. Brains slices were then mounted
on slides and let dry 20 min before being coverslipped
using Vectashield hard set anti-fade mounting (Vector
Laboratories, Burlingame, CA). Sections incubated
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without primary antibody were used as negative con-
trols. Images were acquired using Zeiss 880 confocal
laser microscope (Peabody, MA). Integrated intensity
was quantified using NIH ImageJ (NIH) on raw images,
with background fluorescence subtraction on pre-
defined ROIs.
For Iba-1 VIP-substrate staining (Vector Laboratories,
Burlingame, CA), antigen retrieval was performed using
1X Dako Target Retrieval solution (Agilent Dako, Santa
Clara, CA, USA) in distilled water/0.5% Triton, at 95–
99 °C for 5 min. After washing, endogenous peroxidases
were quenched with 3% H
2
O
2
/0.25% Triton X-100/
1XTBS for 30 min. Sections were incubated in 5% normal
goat serum (Sigma Aldrich) in 1XTBS/0.1% Triton. Pri-
mary polyclonal antibody, anti Iba-1 (Wako Chemicals,
Richmond, VA), was diluted 1:2000 in 1% normal goat
serum in 1XTBS/0.1% Triton and let incubate O/N at
4 °C. Biotin-conjugated goat anti-rabbit secondary anti-
body (SouthernBiotech) were used at 1:2000 in 20% Su-
perBlock (ThermoFisher) in 1X TBS/0.05% Triton, left for
2 h at RT, and lately Streptavidin-HRP (SouthernBiotech)
was incubated for 1h. Staining was visualized using Vec-
tor VIP Substrate (Vector Laboratories) following the
manufacture’s specifications. After washing with distilled
water slides were mounted and coverslipped. Microglia
were imaged on AxioImager Z1 microscope (Zeiss) at 63x
oil and 0.58 μm z-steps to capture 3 ROIs across the
stratum radiatum of the CA1 subfield of the hippocampus
for each animal (5 mice per group, 10 cells per mouse im-
aged: 50 cells per treatment group analysed). The micro-
glia process morphology was categorized with a score
from 0 to 3, following the criteria described by Schafer
et al. [66–68]: 0 (> 15 thin processes with multiple
branches), 1 (5–15 thick processes with branches), 2 (1–5
thick processes with few branches), 3 (no clear processes).
All analyses were performed in blind.
On peripheral organs, histology was performed by His-
toWiz Inc. (histowiz.com) using a Standard Operating
Procedure and fully automated workflow. Samples were
processed, embedded in paraffin, and sectioned at 4 μm.
Immunohistochemistry was performed on a Bond Rx
autostainer (Leica Biosystems) with enzyme treatment
(1:1000) using standard protocols. Slides were stained
with hematoxylin and eosin and anti-NFkb. Bond Poly-
mer Refine Detection (Leica Biosystems) was used ac-
cording to manufacturer’s protocol. After staining,
sections were dehydrated and film coverslipped using a
TissueTek-Prisma and Coverslipper (Sakura). Whole
slide scanning (40X) was performed on an Aperio AT2
(Leica Biosystems).
Primary mouse microglia cultures and uptake experiment
Cultures were prepared from post-natal C57BL/6 mouse
pups at 2 days of age. Whole brains were trypsin
digested and made into a cell suspension. Cells were
seeded in flasks pre-coated with 0.1 mg/ml poly-D-lysine
(Sigma-Aldrich) and maintained in DMEM supple-
mented with 10% heat-inactivated FBS (Gibco) and 1%
Pen-Strep (Gibco). Medium was supplemented with 5
ng/ml Macrophage Colony Stimulating factor (M-CSF)
(Thermo Fisher Scientific) diluted in PBS supplemented
with 0.1% sterile filtered BSA (Sigma-Aldrich). At DIV10
microglia were isolated by orbital shaking at 150 RPM
for 1 h and the supernatant was seeded in 12-well plates
with 300,000 cells per well. Experiments were performed
on the subsequent day. PHF-tau (paired helical fila-
ments) [69] was added to microglia at a concentration of
1μg/ml as determined by total tau ELISA. ScFvMC1 was
added at a concentration of 10 μg/ml. To allow for im-
mune complex formation, PHF-tau and scFvMC1 were
mixed in medium and pre-incubated at 37 °C for 30–45
min prior to addition to cells. Mixing was performed
two times during incubation by repeated manual pipet-
ting. The 2 h incubation was performed in medium with-
out serum. All experiments were performed in triplicate,
with each treatment group in quadruplicate. The
amount of PHF-tau in medium at the end of the experi-
ments was assessed using the same low-tau ELISA previ-
ously described.
Stereotaxic intracranial injection
Intra-hippocampal injections of AAV vectors were per-
formed according to a stereotaxic surgery protocol pre-
viously published [43]. Briefly, under sterile conditions,
3-month-old P301S mice were anesthetized and secured
on a stereotaxic frame (David Kopf instruments,
Tujunga, CA). Mice received bilateral hippocampal in-
jection of AAV preparations using a neuro syringe with
a 33 gauge needle (Hamilton, Reno, NV), using the fol-
lowing coordinates: AP −2.1 from bregma, ML +/−2.0
from bregma, DV −1.8 below dura. Animals were
treated according to the current regulations for the
proper handling of research animals, following an ap-
proved IACUC protocol.
Flow cytometry on adult mice microglia
Microglia was isolated from 6-month-old P301S mice
treated with AAV5-scFv-MC1 and AAV5-null injected
mice. In this experiment, we used an AAV-null con-
struct instead of AAV-eGFP, since our goal was to ascer-
tain the uptake of the scFv by microglia and since eGFP
may interfere with flow cytometry analysis. Mice were
anesthetized and cold PBS-perfused. After dissection,
the forebrain was minced with a Dounce homogenizer
in ice cold HBSS, filtered onto 70 μm cell strainer, and
centrifuged at 300 g for 5 min at 4 °C. Tissue was dissoci-
ated using Neural Dissociation Kit P (MACS Miltenyi
Biotec, Auburn CA) according to the manufacturer’s
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instruction. Myelin debris were removed using Myelin
Removal Beads II (MACS Miltenyi Biotec). Briefly, after
neural dissociation, samples were spun at 300 g for 10
min at 4 °C and incubated 15 min with Myelin Removal
Beads in 0.5% BSA in 1X PBS. Cells suspension was then
loaded onto a pre-washed MACS LS column and placed
in the magnetic field of MACS Separator. The magnetic-
ally labeled myelin was retained within the column while
unlabeled cells run through [70]. Cells suspension, mye-
lin depleted, was then washed twice with FACS buffer
(0.05% BSA, 0.02% sodium azide in 1X PBS) and stained
with Live/Dead-Pacific Blue (Thermo Fisher Scientific).
Surface staining was performed using CD11b-PE and
CD45-APC/Cy7 antibodies (BD Biosciences, Franklin
Lakes, NJ) in order to select microglia from other mono-
cytes. After fixation and permeabilization with BD Cyto-
fix/Cytoperm Fixation/Permeabilization Solution Kit
(BD Biosciences) cells were stained with anti-Myc Tag
Antibody AlexaFluor-647 (Thermo Fisher Scientific).
Debris, doublets and dead cells were excluded using fsc/
ssc, fsc-h/fsc-w and Pacific Blue gates, respectively. BD
CompBeads (BD Biosciences) were used for calibration
of flow cytometer. Samples were analyzed on a BD
LSRFortessa and data processed using FlowJo software
(Treestar).
Tau and anti-scFvMC1 antibodies detection in serum
A detailed protocol was previously published to detect
total tau in serum [71]. Upon sacrifice mice were bled,
samples collected and allowed to clot for 30 min at RT.
After cooling for 15 min, samples were spun at 14,000 g
for 10 min at 4 °C; supernatants were collected and then
re-spun at 14,000 g for 5 min at 4 °C. The final superna-
tants correspond to the serum samples. In order to de-
tect tau in serum, samples were diluted 1:3 in 0.2 M
NaOAc, pH 5.0 and heated at 90 °C for 15 min. After the
heat treatment, samples were allowed to cool at 4 °C for
15 min, and then spun at 15,000 g for 10 min. Superna-
tants were collected and 1 M Tris buffer was added to
neutralize the pH. After diluting 1:2 in 20% Superblock,
samples were loaded on the total tau ELISA (DA31
capture).
In order to detect antibodies directed to the scFvMC1,
96-well plates were coated with purified scFvMC1 at
6μg/ml for at least 24 h. Plates were washed 3X and
blocked for 1 h using StartingBlock (Thermo Fisher Sci-
entific). Plates were washed 5X and 50 μl of sera added
in triplicate at 1:1000 dilution in 20% SuperBlock
(Thermo Fisher Scientific). After 1 h incubation plates
were washed 5X and 50 μl of goat anti-mouse non-
specific IgG HRP-conjugated (SoutherBiotech, Birmin-
gha, AL) was added and incubated for 1 h. Finally, Bio-
Rad HRP Substrate Kit has been used for the detection
and plates were read with Infinite m200 plate reader
(Tecan) at 415 nm.
Statistical analysis
Quantitative data were analyzed using the dedicated
software GraphPad Prism V.6 (GraphPad software Inc.,
CA). Unpaired t test with Welch’s correction was per-
formed when the parametric assumption of normality
(D’Agostino-Pearson omnibus test) was accomplished.
When not, non parametric Mann-Whitney test was per-
formed instead. Statistical significance was set at P<
0.05. Error bars represent the standard error of the mean
(SEM).
Results
ScFvMC1 is detected in the brain homogenates upon
intravenous injection of the purified scFv
We first asked whether scFvMC1 crosses the BBB and
targets the brain [72,73]. After intra-peritoneal (IP) per-
ipheral injection, we were not able to detect brain
scFvMC1 using antibodies directed against Myc or 6-His
(tags present on our scFv construct), via IHC, WB or
ELISA (not shown). We have thus performed a proof-of
concept experiment, testing BBB penetration via retro-
orbital IV injection of 100 μg of purified scFvMC1, either
unlabeled (UNL) or infrared-conjugated (IR), in adult
JNPL3 and P301S mice; a careful comparison was per-
formed between scFvs and native MC1 variants, includ-
ing saline as a negative control. Given the scFv’s short
half-life, mice were sacrificed 2 h post-injection and per-
fused with ice-cold PBS/Heparin. The serum concentra-
tion of both scFvMC1-IR and the native MC1-IR were
calculated in the amount of 50 ng/μl. At termination of
the experiment, scFvMC1 was detected in cortex, HB
and hippocampus (Fig. 1a): its amount was in the range
of 0.1–0.2% of the serum concentration, in line with the
current literature [40,72,74,75], confirming the feasi-
bility of a peripheral approach that relies on a sustained
release of scFv in the circulation.
AAV1-scFvMC1 specifically transduces muscle cells upon
one-time intramuscular injection
Since scFvMC1 efficiently crosses the BBB, we have next
focused on selecting a peripheral tissue to be transduced
by the AAV-scFv construct, to generate a stable source
of antibody, with the goal to circumvent vital organs.
Skeletal muscle is considered an ideal target tissue for
AAV transduction because individual fibers are large,
multinucleated and with minimal cellular turnover [35,
59,76]. Hence, we have selected the AAV1 serotype to
produce a stable muscular niche able to continuously
produce anti-tau scFvMC1 and release it into the circu-
lation to target cerebral tau. In a prevention protocol,
given the different timeline in the development of tau
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pathology in the two mice models selected, 3-month-old
JNPL3 and 2-month-old P301S mice were injected in
the right tibialis anterior muscles with AAV1-CAG-
scFvMC1 and sacrificed 4 months post-injection, at 7 or
6 months of age respectively. To ascertain transduction
of our AAV system, transgene expression in the injected
site was assessed by immunoblotting. Figure 1b shows a
specific band around 30–35 kDa corresponding to scFv-
MC1 expression in the injected tibialis. Other peripheral
organs failed to show expression of scFv-MC1 (Fig. 1c),
confirming targeted local delivery and the high specifi-
city of the AAV1-CAG system. No premature death or
body weight loss were reported throughout the study.
Moreover, no changes in tissues morphology at the time
of sacrifice were detected as assessed by hematoxylin
and eosin (H&E) (Fig. 1d); the inflammatory state, also
unchanged, was assessed using NF-kB staining (Supple-
mentary Fig. 1).
AAV1-scFvMC1 intramuscular injection significantly
decreases the insoluble tau burden in both P301S and
JNPL3 mice
To rigorously quantitate the tau burden, we performed
an extensive biochemical analysis of insoluble, soluble
and oligomeric tau species in brain. We monitored the
tau insoluble burden (a proxy for tau aggregation) in
cortex and hindbrain by low-tau ELISA (Fig. 2,Fig. 3).
As shown in Fig. 2a, we observed a dramatic decrease of
cortical insoluble tau in the P301S cohort upon treat-
ment with scFvMC1: total tau (−70%, **p< 0.01),
pThr231 (−70%, **p< 0.01), pSer202 (−60%, *p< 0.05)
and pSer396/404 (−65%, **p< 0.01). The same
Fig. 1 ScFvMC1 detection and peripheral organs’morphology. aSix-month old mice (JNPL3 shown) were IV-injected with saline, 100 μgof
IRDye-labelled antibodies (scFvMC1-IR and MC1-IR) and unlabeled antibodies (scFvMC1-UNL and MC1-UNL). Two hours post-injection, brain
homogenates from cortex (Ctx), hippocampus (Hip) and hindbrain (HB) were spotted on nitrocellulose and absorbance of the IRDye label
measured. Saline and unlabeled antibodies showed no signal (Sapphire Biomolecular Imager, Azure Biosystems). b,cAAV1-CAG-scFvMC1 was
injected in tibialis muscle of P301S or JNPL3 (JNPL3 shown), mice sacrificed 4 months later and peripheral organs harvested. Tissue lysates were
analyzed for scFvMC1 expression with anti-Myc/tag antibody showing localized expression in the injected site (b, right tibialis, Controls = AAV1-
eGFP) while other peripheral organs did not show scFv signal (c). ScFvMC1: MW around 30 kDa; tubulin is used as housekeeper: MW at 55 kDa. d
Hematoxylin and eosin (H&E) staining was performed on kidney, liver, tibialis muscle and heart: representative images of each organs show no
significant changes in morphology (Controls = AAV1-CAG-eGFP; Bright field microscope, scale bar: 300 μm)
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Fig. 2 Insoluble tau levels are significantly reduced in P301S brains. aIn cortex, insoluble tau levels (INS) were significantly decreased in the
scFvMC1 treated group (n=6)compared to the control cohort (n = 6) (*p< 0.05; **p< 0.01; non parametric Mann-Whitney test). bIn HB,
insoluble tau showed a trend of reduction in the scFvMC1 group (n = 6) compared to the controls (n = 6): total tau (p= 0.0931),
pThr231(p= 0.0931), pSer202 (p= 0.1320), pSer396/404 (p= 0.5887). Graphs are expressed as % Control, and means +/−SEM
Fig. 3 Insoluble tau levels are significantly reduced in JNPL3 brains. aIn the JNPL3 mice, insoluble tau (INS) was significantly reduced in cortex
(ctx) in the AAV1-CAG-scFvMC1 group (scFvMC1) (n= 13) compared to the AAV1-CAG-eGFP injected cohort (Controls) (n = 13). Both total and
phosphorylated tau (three different epitopes pThr231, pSer202 and pSer396/404) were significantly reduced (***p< 0.001; ****p< 0.0001; non
parametric Mann-Whitney test). bTotal and pSer396/404 insoluble tau were unchanged in the JNPL3 hindbrain (HB) in the treated scFvMC1 mice
(n= 13) compared to the controls (n = 13); on the contrary, tau phosphorylated at pThr231, pSer202 exhibited a significant decrease (*p< 0.05;
**p< 0.01, non parametric Mann-Whitney test). Graphs are expressed as % Control, and means +/−SEM
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significant reduction was confirmed in the JNPL3 (Fig.
3a) following a sustained peripheral release of scFvMC1,
both as total tau (−50%, ***p< 0.001) and phosphoryl-
ation at Thr231(−65%,***p< 0.001), Ser202 (−50%,
***p< 0.001) and Ser396/404 (−60%, ****p< 0.0001).
Similar to what we observed in cortex, in the P301S
mice a trend towards reduction was clearly visible in
treated mice’s hindbrains (Fig. 2b): 30% reduction of in-
soluble total tau in hindbrain was detected although this
was not significant at the p= 0.05 level (p= 0.0931); also,
40 and 50% trends to reduction were detected for
pThr231 (p= 0.0931) and pSer202 (p= 0.1320), respect-
ively. The JNPL3 treated mice’s hindbrains exhibited a
significant reduction of pThr231 (−75%, **p< 0.01) and
pSer202 (−65%, *p< 0.05), while total and tau phos-
phorylated at Ser396/404 were not decreased (Fig. 3b).
Tau soluble and oligomeric species’levels show strain
differences upon treatment
We next assessed soluble tau from cortex, hindbrain and
hippocampus homogenates. Biochemical analysis of sol-
uble tau showed differences between the two mice
strains. Analysis of the P301S mice (Fig. 4) showed total
and phosphorylated tau soluble species to be signifi-
cantly reduced in all cerebral regions upon scFvMC1
treatment (Fig. 4a-c; 40–50% reduction,*p< 0.05; **p<
0.01). In contrast, in the JNPL3 mice, we did not observe
any modulation of total, pThr231, pSer202 or pSer396/
404 soluble tau in both cortex and hippocampus (Fig. 5a,
c); surprisingly, in the hindbrain, tau phosphorylated at
Ser202 and Ser396/404 was augmented in the
scFvMC1injected group in JNPL3 mice (Fig. 5b).
Finally, oligomeric/aggregated tau was examined using
a tau-mono-ELISA previously developed in our labora-
tory [65]. This assay, a functional surrogate of insoluble
tau measurement, allows the detection of aggregated tau
species, ranging from dimers to larger aggregates, and
represents a good marker of progression towards the
formation of neurofibrillary pathology in tau transgenic
animal models. We routinely employ this assay for the
measurement of hippocampal aggregated tau. While
JNPL3 mice failed again to show any changes between
controls and treated mice in all brain areas (Fig. 5d),
P301S mice exhibited a significant reduction of oligo-
meric/aggregated tau species in the hippocampus (−
50%, *p< 0.05) and a trend towards reduction in cortex
and HB (Fig. 4d), consistent with the data reported
above for soluble and insoluble tau.
Tau burden changes are not detected by
immunohistochemistry
In addition we performed an immunohistochemical ana-
lysis on JNPL3 (Fig. 6A a-f; Fig. 6B a-f) and P301S brain
slices (Fig. 6Ag-l; Fig. 6Bg-l), by assessing tau
pathology in the CA1 hippocampal pyramidal cell layer
and in the entorhinal cortex (EC). Semi-quantification of
tau phosphorylated at Thr231 and MC1-tau was per-
formed on both CA1 (Fig. 6Ac,i; Fig. 6Bc,i) and EC
(Fig. 6Af,l; Fig. 6Bf,l). No significant changes in im-
munoreactivity were observed, except some trends to-
wards decreased staining in the hippocampal region of
JNPL3 mice (Fig. 6Ac,p= 0.1128; Fig. 6Bc,p=
0.0996). Tau phosphorylation at Ser396/404 and Ser202
(not shown) also failed to show significant reduction
when comparing treated to non-treated animals.
Microglia are not activated, but have the ability to uptake
the tau-scFvMC1 immunocomplex
We next sought to determine whether microglia were
activated in our system. Confocal microscopy was per-
formed on brains from P301S (Fig. 7a, b) and JNPL3
(Fig. 7c, d) mice to assess the immunoreactivity of two
widely studied markers for microglia activation: Iba1
(morphology/activation) and CD68 (reactive/phagocytic).
We observed no difference between controls and
scFvMC1 treated mice and confirmed these data by
semi-quantitative analysis in the stratum radiatum of
both strains. Cortex and HB were imaged and quantified
displaying similar results (not shown).
To examine deeper the microglia activation state, a
morphological assessment was performed (Fig. 8a-c),
using a protocol previously described [66], confirming
that neither P301S or JNPL3 treated mice exhibited any
significant changes in microglia morphology compared
to their respective controls.
Given the peripheral injection and the lack of Fc re-
gion in the scFv structure, we did not expect an inflam-
matory response dependent on the microglia Fc receptor
(FcR) engagement in our model, but we assumed that
the altered functional state of the microglia found in
neurodegenerative disorders and tau mouse models [77–
80] may still facilitate microglia-mediated tau clearance
upon scFv treatment. Hence, to deepen our understand-
ing on the microglia’s role in our system, we first set out
a cell-based proof of concept experiment to determine
whether primary microglia could uptake the tau-
scFvMC1 immunocomplex. Primary microglia were in-
cubated for 2 h with PHF-tau (paired helical filament,
prepared in our lab [69,81]) with and without scFvMC1
(scFv/PHF ratio 10/1) (Fig. 9). In order to work in the
appropriate experimental conditions, PHF-tau and PHF-
tau/scFvMC1 immunocomplex were applied on plastic
plates (−primary microglia) or on microglia seeded
plates (+ primary microglia). Primary microglia showed
an innate ability to uptake PHF from the medium (Fig.
9a,A vs C: 10% reduction, *p= 0.0168), which was en-
hanced by the presence of scFv-MC1: a 20% reduction
(Fig. 9a, C vs D, *p= 0.0137) was detected when co-
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treating the microglia with scFvMC1/PHF compared to
PHF alone. Also, in the co-treatment paradigm we observed
a 25% reduction of PHF in medium (Fig. 9a, B vs D, **p=
0.0019) when working on microglia seeded plates compared
to empty plates. Interestingly, intracellular PHF-tau readily
disappeared, after uptake, from the microglia lysates when
scFvMC1 was present (Fig. 9b, upper panel). Moreover, the
levels of scFvMC1 were unchanged in microglia lysates,
with and without PHF, suggesting an excess of scFv in this
in vitro experiment (Fig. 9b,lowerpanel).
Since we were not able to directly visualize scFvMC1
in the brain upon IM injection of the vectorized anti-
body, to further validate our in vitro data we have uti-
lized adult P301S mice injected intracranially with
AAV5-GFAP-scFvMC1, using the same strategy applied
in our previous study [43]. Confocal microscopy was
performed to co-localize tau and scFvMC1 in microglia
confirming that both phospho-tau (pThr231) and
scFvMC1 (Myc-555) co-localize in Iba1 positive micro-
glia (Supplementary Fig. 2). Using the same experimental
model, we show by confocal microscopy (Fig. 10 A) that
scFvMC1 is detected in Iba1 positive microglia from
treated animals, confirming a role of this cellular popula-
tion in the uptake and clearance of pathological tau. Fi-
nally, adult P301S intracranially injected with AAV5-
GFAP-scFvMC1 were employed to isolate microglia [70,
77,82,83] from whole brain. Following microglia sorting
with flow cytometry (CD11b
high
CD45
low
) (Fig. 10 B, a-
d), cells were permeabilized to detect scFvMC1 inside
the microglia (anti-Myc-647, blue) in the AAV5-
Fig. 4 Soluble and oligomeric tau levels in P301S mice are significantly reduced. a,b,c,dP301S mice were injected at 2 month of age and
sacrificed 4 months post-injection. Levels of total and phosphorylated soluble tau (pThr231, pSer202, pSer396/404) were tested on heat stable
preparations (hsp) from 3 different brain regions, using low-tau ELISA: (a) cortex (ctx), (b) hindbrain (HB) and (c) hippocampus. In all three regions
analyzed, soluble tau levels showed a significant reduction in the AAV1-CAG-scFvMC1 group (scFvMC1) (n=6)compared to the AAV1-CAG-eGFP
cohort (Controls) (n = 6) both as total and phosphorylated levels (*p< 0.05; **p< 0.01 non parametric Mann-Whitney test). (d) Oligomeric/
aggregated tau was measured using the DA9/DA9 mono-ELISA: while a trend towards reduction was found across the cortex and the HB, the
scFvMC1 treated mice (n= 6) exhibited a significant decrease of oligomeric tau in the hippocampus (*p= 0.0303, non parametric Mann-Whitney
test) compared to the controls (n = 6). Graphs are expressed as % Control, and means +/−SEM
Vitale et al. Acta Neuropathologica Communications (2020) 8:126 Page 10 of 19
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scFvMC1 injected mice; no anti-Myc-647 was detected
in the AAV-null injected animals (red) (Fig. 10 B, e).
Overall, our in vitro and in vivo data indicate that, des-
pite the lack of Fc effector function, microglia have the
ability to uptake tau and scFvMC1.
Antibodies directed to the scFvMC1 are detected in the
JNPL3 mice serum
A major concern about the long-term use of antibodies as
a treatment is the generation of neutralizing antibodies
(NAB), which would compromise the therapeutic effect.
We have investigated whether expression of scFv gene in
the body would trigger the production of antibodies
directed against scFv. Upon sacrifice, serum was collected
and processed to test for the presence of scFvMC1 in the
circulation. While we failed to directly detect scFvMC1 in
serum, we were able to detect antibodies directed to
scFvMC1 in the JNPL3 treated cohort (Supplementary
Fig. 3b), similar to what observed in our previous study
upon intracranial injection of AAV5-scFvMC1 [43]. Con-
trarily, the treated P301S mice did not show any detect-
able anti-scFvMC1 in serum (Supplementary Fig. 3a).
Our serological assessment was completed by deter-
mining the tau levels in the circulation, to investigate
the ability of scFvMC1 to export tau from the brain par-
enchyma to the periphery [84]. As shown in
Fig. 5 Soluble and oligomeric tau levels in JNPL3 mice. a,b,c,dJNPL3 mice were injected at 3 months of age and sacrificed 4 months post-
injection. Levels of total and phosphorylated tau (pThr231, pSer202, pSer396/404) were tested on heat stable preparations (hsp) from 3 different
brain regions, using low-tau ELISA: (a) cortex (ctx), (b) hindbrain (HB) and (c) hippocampus. Soluble tau was mostly unchanged in all brain areas
in the AAV1-CAG-scFvMC1group (scFvMC1) (n= 13) compared to the AAV1-CAG-eGFP injected cohort (Controls) (n = 13). Two different
phosphorylation sites, pSer202 (CP13 antibody, early site) and pSer396/404 (PHF1 antibody, late site) were increased in the scFvMC1 treated
group (*p< 0.05, **p< 0.01, non parametric Mann-Whitney test). (d) Oligomeric/aggregated tau was measured using the DA9/DA9 mono-ELISA
showing no modulation in any of the brain areas analyzed (cortex, ctx; hindbrain, HB; hippocampus). Controls are the AAV1-CAG-eGFP injected
mice (n = 13); scFvMC1 corresponds to the AAV1-CAG-scFvMC1 group (n = 13). Graphs are expressed as % Control, and means +/−SEM
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Fig. 6 Phospho-Threonine-231 and MC1-tau immunoreactivity in brain. Brains from P301S and JNPL3 animals were harvested and stained with either
anti tau phospho-Thr231 antibody (A)oranti-MC1-tau(B). (A): a, b, d, e Representative images of CA1 hippocampal cell layer and entorhinal cortex
(EC) stained with anti-pThr231. Control mice received AAV1-CAG-eGFP injection (n = 13). Treated mice were injected with AAV1-CAG-scFvMC1 (n = 13).
(A): c,fSemi-quantification of percentage of area stained by RZ3 shows a trend of reduction in the AAV1-CAG-scFvMC1 injected group, in the CA1
region of hippocampus in the JNPL3 (c,p= 0.1128, unpaired t test with Welch’s correction); no trend to reduction is detected in the entorhinal cortex
(EC) (f,p= 0.3458, unpaired t test with Welch’s correction). (A): g,h,j,kRepresentative images of the CA1 region and entorhinal cortex (EC) from
P301S; pThr-231 staining was performed as above. Control mice received AAV1-CAG-eGFP injection (n = 6), treated mice were injected with AAV1-
CAG-scFvMC1 (n = 6); (A) i, l Semi-quantification of percentage of area stained by RZ3 in P301S mice (i,p= 0.2403; l,p= 0.2251; non parametric Mann-
Whitney test). (B): a,b,d,eRepresentative images of CA1 hippocampal cell layer and entorhinal cortex (EC) stained with anti-MC1 antibody. Control
mice received AAV1-CAG-eGFP injection (n = 13). Treated mice were injected with AAV1-CAG-scFvMC1 (n = 13) (c,f) Semi-quantification of percentage
of area stained by MC1 shows a trend of reduction in the AAV1-CAG-scFvMC1 injected group, in the CA1 region of hippocampus (c,p= 0.0996;
unpaired t test with Welch’s correction); no reduction is detected in the entorhinal cortex (EC) (f,p= 0.9558; unpaired t test with Welch’scorrection)in
JNPL3. (g,h,j,k) Representative images of the CA1 region and the entorhinal cortex (EC) from P301S; MC1 staining was performed as above. Control
mice received AAV1-CAG-eGFP injection (n = 6), treated mice were injected with AAV1-CAG-scFvMC1 (n = 6). (i, l) Semi-quantification of percentage of
area stained by MC1 in P301S mice (i,p= 0.4127; l,p= 0.8413; non parametric Mann-Whitney). (Olympus BH-2 bright field microscope; scale bar:
100 μm). Graphs are expressed as % Control area stained, and means +/−SEM
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Supplementary Fig. 3c, d tau levels did not change upon
treatment in both mice models.
Discussion
The use of antibody fragments has emerged as a promis-
ing approach to target both Aβand tau pathology in
Alzheimer’s disease [32–34,39,40,42–45,85]. We have
previously reported that intracranial administration of
the vectorized anti-tau scFvMC1 was able to reduce dif-
ferent tau species in the JNPL3 transgenic animal model
[43]. This study has set the basis for the development of
a novel therapeutic approach aimed at advancing
Fig. 7 Microglia activation state does not change upon scFvMC1 treatment. For analysis of activation state, sections were immunolabeled for
CD68 and Iba-1 and imaged. aRepresentative image of microglia from P301S controls (n = 6) (upper images) and AAV-scFv-MC1 treated mice
(n = 6) (lower images), in the stratum radiatum: CD68 lysosomal protein (red puncta), Iba1 (green), nuclei stained with DAPI (blue). b
Quantification of Iba-1 and CD68 showed no significant differences between controls and treated mice (p= 0.0931 and p= 0.7771 respectively,
non parametric Mann-Whitney test). cRepresentative image of microglia from JNPL3 controls (n = 13) (upper images) and AAV-scFv-MC1 treated
mice (n = 13) (lower images), in the stratum radiatum. dQuantification of Iba-1 and CD68 showed no significant differences between controls
and treated mice in JNPL3 mice (p= 0.8857 and p= 0.4857 respectively, non parametric Mann-Whitney test). All images were obtained using
Zeiss 880 confocal laser microscope, scale bar: 20 μm. Graphs are expressed as integrated density, and means +/−SEM
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peripheral administration of vectorized anti-tau scFv: in
the present work, we show that IM injection of anti-tau
scFv antibodies has potential for the treatment of
tauopathies.
Few studies [35,45] have previously demonstrated that
intramuscular delivery of an anti-AβscFv gene in an AD
mouse model reduced amyloid deposits and ameliorated
its learning and memory deficits without inducing dis-
cernible inflammation. In line with the current strat-
egies, we have utilized AAV1 to express scFvMC1 in the
striated muscle, to generate a peripheral niche and
achieve long term production of the fragment antibody.
We have extended our investigation to two different
models of tauopathy, reaching significant reduction of
insoluble and soluble tau species, total and phosphory-
lated, with regional and mouse strain differences. The
following considerations are in order.
Detecting scFvMC1 in the brain parenchyma using
antibodies against the Myc or His tags (see construct de-
sign in [43]) has proven challenging when the scFv is
produced in the periphery [35,45], as in the current
work. Contrarily, in our previous publication, where the
scFv was expressed directly in the brain, we were able to
detect and track the diffusion of the scFv by both IHC
and western blot in the 3 brain regions analyzed (hippo-
campus, cortex, hindbrain) [43]. We thus hypothesized
that the local brain concentration of the peripherally
generated scFv would not be enough for detection.
Using this systemic approach we also failed to detect
scFv in serum: it is plausible that, together with the de-
tection limits of the assay employed, a certain amount of
recombinant antibody might get misfolded and de-
graded, explaining why we can’t visualize scFv in serum.
When we conjugated the scFvMC1 to an IR dye and
injected 100 μg of it by IV route (less than half the dose
normally injected in anti-tau passive immunotherapy ex-
periments in mice, i.e. 10 mg/kg), we were able to detect
the compound in the brain, and similarly for the native
MC1 antibody. Importantly, the ratio between the blood
and brain parenchyma concentrations are in line with
what seen by others [72,74,86,87] for both the native
and scFv antibody. Hence, these data suggest that our
scFv is able to cross the BBB and reach its target, justify-
ing its use in a systemic delivery paradigm. Consistent
with this proof-of-concept experiment, the efficacy data
show that peripherally generated scFvMC1 can reach the
brain parenchyma to modulate tau levels, with no per-
ipheral leakage of the transgene expression, conferring
to this approach a relevant translational advantage.
Previous studies from our laboratory and others [14,
16] have investigated the effect of tau passive immuno-
therapy using peripherally injected native MC1, in both
JNPL3 and P301S mice. Comparing the present study to
the previous ones is not trivial, given the different nature
of the antibodies (full antibody vs scFv), the variability of
the mice strains and the delivery routes employed. One
important finding in the present work is the highly sig-
nificant decrease of insoluble total and phosphorylated
tau in cortex, with similar response in P301S and JNPL3
mice. P301S soluble tau was also reduced across the
Fig. 8 Microglia morphology is unchanged upon treatment.
Microglia processes morphology was assessed in both P301S and
JNPL3 animals. aRepresentative images of Iba-1 positive microglia
(stratum radiatum of the CA1 subfield of the hippocampus): the
processes morphology was scored as 0 (> 15 thin processes with
multiple branches), 1 (5–15 thick processes with branches), 2 (1–5
thick processes with few branches), 3 (no clear processes). b
Microglia from P301S mice did not show any significant
morphological changes comparing controls to treated mice. Each
single point represents a single cell (n= 50 cells per group, p=
0.5671, non parametric Mann Whitney test). cNo significant changes
in microglia morphology were detected in the JNPL3 cohort (n = 50
cells per group, p= 0.9628, non parametric Mann Whitney test).
Graphs are expressed as arbitrary unit (a.u.) and means +/−SEM.
(AxioImager Z1 microscope, Zeiss; 63x oil and 0.58 μm z-steps)
Vitale et al. Acta Neuropathologica Communications (2020) 8:126 Page 14 of 19
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

brain, while oligomeric/aggregated tau was reduced
mostly in the hippocampus. On the contrary, we did not
observe any reduction of soluble and oligomeric species
in the JNPL3 mice, and detected instead an increase of 2
phospho-tau epitopes in the hindbrain: we have no clear
explanation at this point for the increase of soluble
pSer202 (early p-tau epitope in AD) and pSer396/404
(late p-tau epitope in AD) tau in the JNPL3 HB, which is
opposite to the other findings. Semi-quantitative analysis
of RZ3 and MC1 immunohistochemistry staining on
brain slices did not confirm the reduction of soluble tau
detected biochemically by ELISA, in the P301S model,
which is more sensitive to changes in soluble and insol-
uble tau. Taken together, these data point to regional
and qualitative (soluble vs insoluble) differences in tau
clearance in both mice models.
Indeed, mice strains may vary in the quantitative and
topographic expression of the tau transgene (by virtue of
promoters and copy numbers), rate of deposition of tau
pathology and tau phosphorylation, inflammation/react-
ive changes and BBB permeability to the scFv. As previ-
ously reported, in contrast to the JNPL3 model, the
P301S mice accumulate detectable levels of hyperpho-
sphorylated tau species (64-kDa) in soluble as well as in-
soluble fractions [16,63]. The precise reason for this
difference between the pathological tau accumulation in
the two models is unclear but may explain some discrep-
ancy in the effect of anti-tau immunotherapy.
The use of antibodies to tackle tau pathology is fascin-
ating, but may carry consequences in terms of eliciting
an inflammatory response, by activation of immune cells
via Fc receptor. In our system, the absence of the Fc re-
gion should prevent such occurrence. Indeed, we have
not found any signs of microglia activation, nor we have
found signs of peripheral tissue damage from the AAV-
scFvMC1 transduction, or increased pro-inflammatory
cytokines levels in serum. Hence, so far, our strategy ap-
pears to be safe. However, the host immune system
could pose some limitations, such as cellular immune re-
sponses to AAV vector and/or transgene. In this respect,
the AAV field is moving into developing strategies to
overcome immunogenicity produced by re-
administration [88]. In our study, we have detected anti-
bodies directed to scFvMC1 in the JNPL3 cohort, which
did not prevent efficacy, in line with what reported earl-
ier [43].
Depending on the administration route, the dose, and/
or the AAV serotype, gene transfer can result in a dele-
terious immune response [89,90] or tolerance [61,91,
92]. Two studies have shown that AAV1 IM injection
can lead to regulatory T cells (Tregs) infiltration in
injected muscles in AATD (Alpha-1 Antitrypsin
Fig. 9 Microglia uptake phosphorylated tau in vitro,facilitated by scFvMC1. (a) Primary mouse microglia (P2 C57Bl/6 J pups) were treated in vitro
for 2 h with PHF-tau +/−scFvMC1 (scFv/PHF ratio of 10/1). Total tau ELISA. Column A:PHF levels are expressed as % of starting PHF
concentration measured after incubation on cell-free plates (−primary microglia); column B:amount of PHF in medium upon combination with
scFvMC1, on cell-free plates (−primary microglia); column C and column D:PHF levels on microglia seeded plates (+ primary microglia), with or
without scFvMC1. A vs C (*p< 0.05, unpaired t test with Welch’s correction); C vs D (*p= 0.0137, unpaired t test with Welch’s correction); B vs D
(**p= 0.0019 unpaired t test with Welch’s correction). Data are collected from three different experiments, with treatments run in quadruplicates.
Graphs are means +/−SEM. (b, upper panel) Representative immuno-blotting (PHF1 antibody: anti-pSer396/404) of PHF-tau in the corresponding
microglia lysates; (b, lower panel) scFvMC1 expression verified using anti Myc-tag antibody. NT is non treated microglia; PHF is microglia treated
with PHF-tau; PHF + scFv is microglia co-treated with PHF-tau and scFvMC1; scFv is microglia treated with scFvMC1
Vitale et al. Acta Neuropathologica Communications (2020) 8:126 Page 15 of 19
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Deficiency) and LPLD (Lipoprotein Lipase Deficiency)
patients [61,91]. Gernoux et al. [92] have recently corre-
lated results obtained in monkeys to those obtained with
patients and further demonstrated that tolerance and
persisting transgene expression after AAV1 gene transfer
in muscle is mediated by Tregs and exhausted T cells.
These findings support the IM approach proposed in the
present study and its translational potential for humans.
One last mechanistic aspect was investigated: the role
of microglia in clearing tau after scFvMC1 administra-
tion. Luo et al. [93] have reported that murine microglia
rapidly internalize and degrade hyperphosphorylated
pathological tau isolated from AD brain tissue in a time-
dependent manner in vitro, showing that the anti-tau
monoclonal antibody MC1 had the ability to enhances
microglia-mediated tau degradation in an Fc-dependent
manner. Despite the lack of Fc in our scFvMC1, we
wanted to investigate this mechanistic aspect, assuming
that the microglia could still participate in the removal
of the immunocomplex in our model. Thus, we took
both an in vitro and in vivo approach to determine if
microglia are able to uptake PHF (hyperphosphorylated
aggregated tau) with or without the help of scFv. We
found that in vitro, cultured microglia were able to up-
take PHF-tau (confirming the previous study [93]) and
scFvMC1 had the ability to enhances this process; fur-
thermore, scFvMC1 accelerated the degradation of PHF
tau in these cells. In vivo, we have been able to co-
localize tau (anti-pThr231), scFvMC1 (anti-Myc) and
microglia cells (anti-Iba1) after intracranial injection of
AAV-scFvMC1. It is plausible that multiple mechanisms
contribute to the clearance of tau upon scFv
Fig. 10 Microglia uptake scFvMC1 in vivo.(A) P301S were injected intracranially in the CA1 quadrant of the hippocampus using AAV5-GFAP-
scFvMC1. Upper panels: Representative confocal images of the cortex: scFvMC1 (Myc, red) co-localizes within the microglia (Iba1, green); nuclei
stained with DAPI (blue). Lower panels: higher magnification to better visualize scFvMC1 in microglia positive cells (Zeiss880 confocal laser
microscope; upper panels, scale bar: 20 μm; lower panels, scale bar: 10 μm). (B) Flow cytometry on microglia isolated from adult P301S mice
intracranially injected with AAV5-GFAP-scFvMC1 or AAV5-null (a-c) Gating strategy (live, singlets) for subsequent selection of microglia. (d) Gating
strategy to isolate microglia from other monocytes. Representative plot showing microglia population: CD11b
high
and CD45
low
; near-complete
absence of macrophages: CD11b
high
and CD45
high
.(e) Microglia extracted from P301S mice, treated (blue) or not (red) with scFv-MC1: upon
permeabilization, anti-Myc-647 detects scFvMC1 in microglia of treated mice (blue)
Vitale et al. Acta Neuropathologica Communications (2020) 8:126 Page 16 of 19
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

immunotherapy and that microglia uptake is one of the
routes the brain uses to remove pathological tau. Future
experiments will investigate whether microglia or other
CNS or peripheral cell types participate in the uptake of
scFvMC1 and tau using receptors other than the FcR,
i.e. toll-like receptors (TLRs), C-X3-C motif chemokine
receptor 1 (CX3CR1), pattern-recognition receptors
(PRRs), scavenger receptors, and complement protein
C1q receptor (C1q-R).
Conclusions
To our knowledge, this work provides the first descrip-
tion of vectorized anti-tau scFv exerting an effect in the
brain upon intramuscular injection. More studies are
planned to investigate whether the highly significant re-
duction of insoluble tau gained in both animal models
will also ameliorate their behavioral phenotype. Also,
from a clinical perspective, particularly attractive would
be the ability to not only express the scFv but to switch
it on or off at will, adding a layer of exogenous control
to improve safety. Among the currently available in-
ducer/repressor systems permitting control over gene
expression, the tetracycline (Tet)-dependent system is by
far the most advanced and most widely used, and has
been already tested in association with AAV1 and locor-
egional muscle gene transfer in non-human primates
showing no humoral or cellular responses against the
transgene [94]: we will certainly explore this avenue in
our system.
In addition, our work opens the field to future studies
reaching beyond scFv, to test engineered antibodies with
multiple specificities and targets in the brain, using a
simple and translatable approach.
Overall, given the limitations linked to conventional
immunotherapy in neurodegenerative diseases, this work
demonstrates the efficacy and the advantages of using
intramuscular injection of vectorized scFv to target tau,
and its relevant translational features, suggesting poten-
tial applications to other brain proteinopathies.
Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s40478-020-01003-7.
Additional file 1 Supplementary figure 1: Inflammatory status in
peripheral organs. NF-kB immunoreactivity, marker of activated proinflam-
matory pathways, was evaluated on kidney, liver, tibialis muscle and
heart: representative images of each organs do not show differences be-
tween controls and AAV1-scFvMC1 treated mice (Controls = AAV1-CAG-
eGFP; Bright field microscope, scale bar: 300 μm).
Additional file 2 Supplementary figure 2. Phospho-tau and scFvMC1
co-localize in microglia. Representative confocal image of the stratum
radiatum from P301S mice injected with AAV5-GFAP-scFvMC1. Astrocytes
actively express scFv-MC1 (Myc-red); Iba1 positive microglia (green)
shows co-localization of scFvMC1 (myc-red) and pTau (pThr231; blue):
merge purple (white arrows). Zeiss880 confocal laser microscope: merge
image is 2x crop of 40X magnification; scale bar: 20 μm.
Additional file 3 Supplementary figure 3 Anti-scFvMC1and tau in
serum. (a, b) Antibodies anti-scFvMC1 were assessed using a specific im-
mune sorbent assay: JNPL3 mice receiving the scFvMC1 exhibited a sig-
nificant increase of anti-scFvMC1 in serum (***p= 0.0005, non parametric
Mann-Whitney test) compared to the controls; no anti-scFvMC1 were de-
tected in the P301S animals; (c,d) total tau levels were measured in
serum at the end of the 4 month treatment: no significant changes were
identified in both transgenic models, P301S and JNPL3. Graphs are
expressed as Arbitrary Units (a,b) or % Control (c,d), and means +/−
SEM.
Abbreviations
Ab: Antibody; Aβ: Amyloid-beta; AAV: Adeno-associated viral vector;
AD: Alzheimer’s disease; BBB: Blood-brain barrier; CNS: Central nervous
system; Ctx: Cortex; EC: Entorhinal cortex; HB: Hind brain;
Hippo: Hippocampus; IM: Intramuscular; IV: Intravenous; LV: Lentiviral vector;
mAbs: Monoclonal antibodies; PHF: Paired helical filaments; RD: Stratum
radiatum; scFv: Single chain variable fragment; scFvMC1: Single chain variable
fragment MC1; Tregs: Regulatory T cells; V
H
: Variable heavy chain; V
L
: Variable
light chain
Acknowledgements
We thank Dr. Peter Davies for providing tau antibodies and PHF-tau. We would
like to thank Dr. Czeslawa Kowal for helping with retro-orbital injections.
Ethic approval and consent to participate
All experiments were conducted under the institutional guidelines and were
approved by the Institutional Animal Care and Use Committee at The
Feinstein Institutes for Medical Research, Northwell Health.
Authors’contributions
FV performed experiments, analysed data and helped preparing the
manuscript; JO performed experiments and helped preparing figures; BTV
analysed and interpreted data; PM interpreted experiments and edited the
manuscript; LG designed the overall project, analysed data, interpreted
results and prepared the manuscript; CD designed the overall project,
performed experiments, analysed data, interpreted results and prepared the
manuscript. The authors read and approved the final manuscript.
Funding
This study was supported by the National Institute of Health (NIH) grant
1R56AG055479–01 to C. d’Abramo.
Availability of data and materials
The datasets used and/or analyzed during the current study are available
from the corresponding authors upon reasonable request.
Consent for publication
Not applicable.
Competing interests
The authors declare no conflict of interest.
Author details
1
Institute of Molecular Medicine, The Litwin-Zucker Center for Alzheimer’s
Disease & Memory Disorder, The Feintein Institutes for Medical Research,
Manhasset, NY, USA.
2
Donald and Barbara Zucker School of Medicine at
Hofstra/Northwell, Hempstead, NY, USA.
3
Institute of Molecular Medicine,
Center for Autoimmune and Musculoskeletal Disease, The Feinstein Institutes
for Medical Research, Manhasset, USA.
4
Northwell Health Neuroscience
Institute, Northwell Health System, Manhasset, NY, USA.
Received: 15 May 2020 Accepted: 27 July 2020
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