Journal of Neuroinflammation(2024) 21:196
Lee et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03192-7

Open Access

RESEARCH

Enhanced phagocytosis associatedwith multinucleated microglia via Pyk2
inhibition in an acute β‑amyloid infusion model
Ji‑Won Lee1*, Kaito Mizuno1,2, Haruhisa Watanabe3,6, In‑Hee Lee4, Takuya Tsumita5, Kyoko Hida5,
Yasutaka Yawaka2, Yoshimasa Kitagawa6, Akira Hasebe1, Tadahiro Iimura3 and Sek Won Kong4,7Abstract
Multinucleated microglia have been observed in contexts associated with infection, inflammation, and aging.
Though commonly linked to pathological conditions, the larger cell size of multinucleated microglia might enhancetheir phagocytic functions, potentially aiding in the clearance of brain debris and suggesting a reassessmentof their pathological significance. To assess the phagocytic capacity of multinucleated microglia and its implications
for brain debris clearance, we induced their formation by inhibiting Pyk2 activity using the pharmacological inhibitor
PF-431396, which triggers cytokinesis regression. Multinucleated microglia demonstrate enhanced phagocytic function, as evidenced by their increased capacity to engulf β-amyloid (Aβ) oligomers. Concurrently, the phosphorylationof Pyk2, induced by Aβ peptide, was diminished upon treatment with a Pyk2 inhibitor (Pyk2-Inh, PF-431396). Furthermore, the increased expression of Lamp1, a lysosomal marker, with Pyk2-inh treatment, suggests an enhancementin proteolytic activity. In vivo, we generated an acute Alzheimer’s disease (AD) model by infusing Aβ into the brainsof Iba-1 EGFP transgenic (Tg) mice. The administration of the Pyk2-Inh led to an increased migration of microglia
toward amyloid deposits in the brains of Iba-1 EGFP Tg mice, accompanied by morphological activation, suggestinga heightened affinity for Aβ. In human microglia, lipopolysaccharide (LPS)-induced inflammatory responses showedthat inhibition of Pyk2 signaling significantly reduced the transcription and protein expression of pro-inflammatory
markers. These results suggest that Pyk2 inhibition can modulate microglial functions, potentially reducing neuroinflammation and aiding in the clearance of neurodegenerative disease markers. This highlights Pyk2 as a promisingtarget for therapeutic intervention in neurodegenerative diseases.
Keywords Protein Tyrosine Kinase 2B, Microglia, Multinucleation, Phagocytosis, β-amyloid, Alzheimer’s disease*Correspondence:
Ji‑Won Lee
jwlee@den.hokudai.ac.jp
1
Microbiology, Department of Oral Pathobiological Science, Facultyand Graduate School of Dental Medicine, Hokkaido University, Kita13
Nishi7, Kita‑Ku, Sapporo 060‑8586, Japan
2
Dentistry for Children and Disabled Persons, Department of Oral
Functional Science, Faculty of Dental Medicine, Hokkaido University,
Kita13 Nishi7, Kita‑Ku, Sapporo 060‑8586, Japan3
Department of Pharmacology, Faculty and Graduate School of Dental
Medicine, Hokkaido University, Kita13 Nishi7, Kita‑Ku, Sapporo 060‑8586,
Japan
4
Computational Health and Informatics Program, Boston Children’s
Hospital, Boston, MA 02215, USA

5

Department of Vascular Biology and Molecular Pathology, Facultyand Graduate School of Dental Medicine, Hokkaido University, Kita13
Nishi7, Kita‑Ku, Sapporo 060‑8586, Japan
6
Oral Diagnosis and Medicine, Department of Oral Pathobiological
Science, Faculty of Dental Medicine, Hokkaido University, Kita13 Nishi7,
Kita‑Ku, Sapporo 060‑8586, Japan
7
Department of Pediatrics, Harvard Medical School, Boston, MA 02115,
USA© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as longas you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article orparts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicatedotherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To
view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Lee et al. Journal of Neuroinflammation(2024) 21:196

Background
Alzheimer’s disease (AD) is a complex neurodegenerative disorder characterized by progressive cognitive
decline, memory loss and impaired executive function[1]. Representative hallmark of AD is the accumulation of
β-amyloid (Aβ) plaque in the brain [2]. Aβ peptides arederived from the cleavage of amyloid precursor protein(APP) by enzymes such β-secretase and ɣ-secretase [3].
These Aβ peptides aggregate to form insoluble plaques,
which are neurotoxic and disrupt neuronal function. As
such, the genes involved in the production and process
APP, including APP, PSEN1, PSEN2 (components of theɣ-secretase complex), and BACE1 (β-secretase), havebeen consistently reported as genetic risk factors forfamilial AD [4]. However, small molecules and biologics
targeting β-secretase or ɣ-secretase complex have notbeen successful in reduced Aβ production [5–10]. Therefore, alternative therapeutic strategies to effectively prevent Aβ accumulation are urgently needed to prevent anddelay the progression of AD.
Phagocytosis is the process by which immune cells
engulf and digest foreign particles, pathogens, and cellular debris. In the brain, microglia play a central role inphagocytosing Aβ plaques [11]. Enhancing the clearanceof Aβ plaque through microglial phagocytosis represents
a potential therapeutic strategy to mitigate Aβ-induced
neurotoxicity and slow disease progression in AD [12].
Microglia phagocytosis of Aβ can also modulate neuroinflammatory responses [13]. Efficient clearance of Aβ by
microglia may dampen neuroinflammation by reducing
the accumulation of Aβ plaques and the release of proinflammatory cytokines, thereby attenuating neuronaldamage and preserving cognitive function.
Microglia, the tissue-resident macrophages derived
from yolk-sac myeloid progenitors, migrate to the central nervous system (CNS) during fetal brain development, playing a crucial role in brain development,
homeostasis and responding to pathological conditions [14]. The activation and functions of microglia
are closely linked to various neurodegenerative diseases, including Alzheimer’s disease (AD) [15–17],
Parkinson’s disease (PD) [18, 19], and multiple sclerosis (MS) [20–22]. As innate immune cells of the CNS,
once activated, microglia participate in the clearanceof misfolded proteins, debris, and pathogens, thereby
mitigating neuroinflammation and preserving neuronal
integrity [23, 24]. The inefficient phagocytic activity of
microglia with aging may lead to the formation of Aβplaques, neuronal dystrophy characterized by tau accumulation, and neuroinflammation, which collectively
represent the core pathologies of AD [25, 26]. Microglial activation and phagocytosis are intricately linked

Page 2 of 19

to rapid cytoskeletal remodeling and focal adhesion,
which encompasses membrane ruffling and extension[27]. The cytoskeleton facilitates the bringing togetherof surface receptors and their substrates to regulate signal transduction, thereby transitioning microglia froman inactive state to an active state [28]. Hence, the capability for rapid morphological change in response todiverse stresses is essential for microglia phagocytosis.
Genetic studies of late-onset AD (LOAD) have identified various risk factors [29, 30]. The Protein Tyrosine
Kinase 2 Beta (PTK2B) gene, which encodes protein
tyrosine kinase (Pyk2), a non-receptor tyrosine kinase
and a member of the focal adhesion kinase (FAK)
family, has been identified as a genetic risk factor ingenome-wide association studies (GWAS) for LOAD
[29]. Indeed, Pyk2 is expressed in diverse brain cells of
AD patients [30–35]. Unlike FAK, which is ubiquitously
expressed across various cell types, Pyk2 expression is
predominantly found in cells of the CNS and hematopoietic cells [36, 37]. Pyk2 is activated by various
extracellular signals including cytokines, intracellular­Ca2+ concentration, and integrin-mediated cell adhesion [38] and has diverse physiological and pathologicalroles in cell adhesion [39], migration [40], inflammatory responses [41, 42], tumor invasiveness [43] andneuronal plasticity [44]. Although PTK2B is recognized
for its expression in neuronal cells [45, 46], recent studies have highlighted its significant role in microglia[47]. Thus, understanding the functional role of Pyk2 inmicroglia and its impact on AD progression is crucial
for uncovering novel pathophysiological mechanisms
underlying AD.
In the current study, we found that the pharmacological inhibitor, PF-431396, targeting Pyk2, leads tomicroglial multinucleation, irrespective of infection or
inflammation responses. These multinucleated microglia exhibit enhanced phagocytosis and lysosomal
activity compared to their mononuclear counterparts.
To this end, we established an acute AD mouse modelby directly infusing Aβ into the intracerebroventricular(ICV) region of brain using brain infusion kit in Iba-1
EGFP Tg mice. This approach enables efficient observation of microglial response to Aβ, circumventing
the several months typically required for genetic AD
mouse models. Administration of PF-431396 not only
enhanced microglial activity but also improved phagocytosis, aiding in the removal of Aβ oligomers fromthe brains of Iba-1 EGFP Tg mice. Our findings indicate that pharmacological inhibition of Pyk2 signaling
could offer potential in treating or delaying the onset
and progression of neurodegenerative disorders suchas AD, through the modulation of microglial phagolysomal activity.

Lee et al. Journal of Neuroinflammation(2024) 21:196

Materials and methods
Preparation of Pyk2‑Inh and β‑amyloid

Pyk2 inhibitor (Pyk2-Inh, PF-431396, CAS no. 71790629-1) was purchased from TOCRIS Biosciences/Biotechne (Minneapolis, MN) and dissolved in dimethyl
sulfoxide (DMSO) (Sigma, MO), then diluted to corresponding concentrations. All the control group wastreated with an equivalent percentage of DMSO. The
final concentration of DMSO did not exceed 0.01%.
Murine recombinant CSF1 was obtained from PeproTech (Cat. no. AF-315-02, Japan). Lyophilized synthetic unlabeled β-amyloid peptide (1–42, Abcam Cat.
no. ab120959) were dissolved in 1% N
­ H4OH (Anaspec,
Cat. no. As-61322) at 1 mg/mL and vortexed for 30 s
according to the manufacturer’s protocol [48, 49]. The
peptides were then lyophilized and stored at – 80 ℃
until use. For experiments, the peptides were reconstituted in 10 mM sodium phosphate buffer (pH 7.4) at aconcentration of 1 mg/mL. β-amyloid (1–42) H­ iLyte™
Fluor555-labeled peptide (Anaspec Inc., Cat. no.
AS-60480-01) was reconstituted in a buffer containing
50 mM Tris (pH 7.4) and 0.1% N­ H4OH and stored at− 80 ℃ until use according to the manufacturer’s protocol. The peptide was diluted with a PBS at a working concentration of 1 mg/mL. For brain injection, theAβ peptide was diluted in artificial cerebrospinal fluid(ACSF, TOCRIS, Cat. No. 3525), incubated at 37 ℃ for
10 min, and then placed on ice before use [50].
Animals

Iba-1 EGFP Tg mice were obtained from RIKEN BRC(Japan). Mice were housed under controlled lighting
conditions (daily light, 07:00–21:00) and were maintained under specific pathogen-free conditions. A totalof 6 mice were randomly divided into two groups (n = 3
per group) with a weight deviation of less than 5% andcompared pharmacological phenotypes of Pyk2-Inh in
7-week-old Iba-1 EGFP Tg mice (littermates, female).
Cell cultures

The mouse microglia cell line MG6 (Riken BRC, Japan)
was cultured in Dulbecco’s Modified Eagle Medium(DMEM—high glucose, Gibco, Life Technologies Corporation, CA) supplemented with 10 μg/mL insulin,
0.1 mM 2-mercaptoethanol (2-ME, Sigma) and 10%
fetal bovine serum (FBS) at 37 ℃ in a humidified atmosphere of 5% ­CO2. The human microglia cell line HMC3(ATCC, CRL-3304, VA) was cultured in Minimum
Essential Medium Eagle (MEME, Sigma) supplemented
with non-essential amino acid, sodium pyruvate and
10% FBS at 37 ℃ in a humidified atmosphere of 5%

Page 3 of 19­CO2. The medium was changed every 3 days until
harvested.
Cell proliferation assay

Cells were seeded in a 96-well plate (1 × ­104 cells/well)and cultured at 37 ℃ for 24 h. Cell proliferation wasdetermined by a 3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay with Cell Count Kit-8(Dojindo, Japan) according to the manufacturer’s protocol. Measurement was carried out in absorbance at
570 nm with a microplate reader (Bio-Rad, Japan). The
cell viability was standardized to the relative control as apercentage.
Total RNA was extracted from cells using ­RNAeasy®
Mini kit (Qiagen, Hilden, Germany) according to the
manufacturer’s protocol. RNA was quantified using aspectrophotometry (DeNovix, DS-11, Japan). The template RNA was used to generate complementary DNA(cDNA) via a Reverse Transcription Kit according to the
manufacturer’s instructions on ice (­QuantiTect®, Qiagen). Quantitative real-time RT-PCR was carried out
with DNA probes PowerUp SYBR Green Master Mix(Thermo Fisher, CA). The primer sequences (Eurofins
Genomics, Japan) were listed in Supplemental Table 1. To
normalize the amount of RNA, the expression of Gapdh
and RPLP0 were chosen as the endogenous control formouse and human, respectively. Expression of target
mRNA was calculated from 2Δ Ct values.
Quantitative RT‑PCR (qPCR)

Immunoblotting analysis

Total protein was extracted from cells using ice-cold
radio immunoprecipitation assay buffer (RIPA, Abcam)
containing protease and phosphatase inhibitor cocktail(Sigma) and incubated on ice for 10 min. Cell lysis wascentrifuged at 12,000 rpm at 4 ℃ for 10 min and supernatants were separated by 7.5% Mini-PROTEAN TGX Gels
(Bio-Rad, USA) electrophoresis and transferred to polyvinylidene fluoride membranes (PVDF, Bio-Rad). The
membranes were blocked with 5% bovine serum albumin(BSA) in Tris-Buffered Saline with Tween 20 (TBST) (pH7.6) (Takara Bio, Japan) for 30 min at room temperature
with gentle shaking, followed by incubation with primary
antibodies overnight at 4 ℃. The primary antibodies
detected immuno-blotting were listed as follows: p-Pyk2(Anti-rabbit, Cell Signaling Technology #3291, 1:1000),
Pyk2 (Anti-rabbit, Cell Signaling Technology #3292,
1:1000), pFAK (Anti-rabbit, Cell Signaling Technology#3283, 1:1000), FAK (Anti-Rabbit, Cell Signaling Technology #3285, 1:1000), p-ERK (Anti-Rabbit, Cell Signaling Technology #4370, 1:1000), ERK (Anti-Rabbit, Cell
Signaling Technology #9102, 1:1000), p-Src (Anti-rabbit,

Lee et al. Journal of Neuroinflammation(2024) 21:196

Cell Signaling Technology #6943, 1:1000), Src (Antirabbit, Cell Signaling Technology #2108, 1:1000), Mannose receptor (Anti-mouse, Abcam ab64693, 1:1000),
Lamp-1 (Anti-rabbit, Abcam ab24170, 1:1000), p-NF-ϰB(Anti-rabbit, Cell Signaling Technology #3033, 1:1000),
NF-ϰB (Anti-rabbit, Cell Signaling Technology #8242,
1:1000), β-actin (Anti-mouse, Wako, 010-27841, 1:1000)
and Tubulin (Anti-rat, Abcam ab6160, 1:1000). The
membranes were washed 3 times with 1 × TBST for
10 min, then added secondary antibodies (Anti-Rabbit,
EnVision+System- HRP Labelled Polymer Dako, USA) or(Anti-mouse, EnVision+System-HRP Labelled Polymer,
Dako, USA) for 1.5 h at room temperature with gentle
shaking. The protein bands were visualized using a Clarity™ Western ECL Substrate (Bio-Rad, US) and exposed
to an iBright CL1500 Imaging System (Invitrogen).
Results were standardized using housekeeping protein.
Phagocytosis of E. coli BioParticles

Microglia treated with Pyk2-Inh for 24 h were sorted
into mononucleated and multinucleated cells using a fluorescence-activated cell sorter (FACS, BD Biosciences).
Briefly, MG6 cells were stained with Hoechst 33,342 for
5 min before being loaded into the FACS. Then, cells withor without Pyk2-Inh were suspended in ­autoMACS®
Running Buffer (Myltenyi Biotec, CA). Sorting was basedon the size difference between mononucleated cells(small) and multinucleated cells (large). The small cell
fraction and the large cell fraction were distinguishedusing forward scatter-area (FSC-A) and side scatterarea (SSC-A) and then sorted by BD F
­ ACSAriaTMII Cell
Sorter respectively. Sorted MG6 cells were seeded at adensity of 5 × ­103 cells/well in a plate. pHrodo Red E. coli
BioParticles (Invitrogen), at a concentration of 10 μg/mL,
were added to the MG6 cells, and the culture was incubated at 37 ℃ for 30 min to 4 h to allow phagocytosis
and acidification until to reach their maximum level. At
every indicated time points, fluorescence was measured
using fluorescence plate reader (PerkinElmer Wallac1420­ARVOTMSFL1) with excitation 560 nm and emission
585 nm wavelength. For imaging analysis, MG6 cells
were seed in a μ-slide 8-well chamber (ibidi, Germany).
After 30 min of exposure to BioParticles, cells were fixedwith 10% formalin and subjected to immunocytochemistry. Particles were quantified by IMARIS software.
To measure the lysosomal activity, we utilized the ­DQ™
Green bovine serum albumin (BSA) (Invitrogen, cat. no.
D12050). The conjugated BODIPY dye (self-quenchedstatus) produces fluorescence (dequenching status)
through intracellular degradation of fluorescein derivatives by proteolysis activity. MG6 cells were seeded at 1
Lysosomal activity

Page 4 of 19

× ­105 cells/well in a 96-well black clear bottom plate andcultured in medium containing CSF1 (100 ng/mL). After
24 h, Pyk2 inhibitor was added at varying concentrations(0, 500, 1000 nM). Following another 24 h, DQ Green
BSA was administered at a concentration of 20 μg/mL,
and fluorescence was measured using fluorescence plate
reader (PerkinElmer Wallac1420 A­ RVOTMSFL1) withexcitation 355 nm and emission 460 nm wavelength after
3 h.
Immunohisto − and cytochemistry analysis

MG6 cells were treated with or without Pyk2-Inh(1000 ng/mL) in the presence of Aβ at a concentration of
500 ng/mL. For immunostaining, cells were fixed with 4%paraformaldehyde in PBS for 10 min, permeabilized with
0.1% Triton X-100/PBS for 30 min, followed by blocking with 0.1% BSA (Sigma) for 30 min. Cells were stainedwith the indicated specific antibodies. The primary antibodies detected immuno-cytochemistry and -histochemistry were listed as follows: Iba-1 (Anti-Rabbit, Wako,
019-19741, 1:200), Tubulin (Anti-rat, Abcam ab6160,
1:100), Pericentrin (Anti-rabbit, Abcam, ab4448, 1:100)
and IntegrinαV (Anti-mouse, Abcam, ab16821, 1:100).
To quench the fixation agent and eliminate any free aldehyde groups that could non-specifically bind antibodies, sections or cells were treated with 0.1 M Tris buffer.
Immunofluorescence images were captured using a laser
confocal scanning microscope (A1-ECLIPSE Ti2-E,
Nikon, Japan) equipped with an oil immersion objective
lens, PLAN APO λD 60X (NA = 1.42). Prior to performing live-cell imaging, cells cultured in the chamber slidewere incubated with Hoechst 33,342 (ThermoFisher Scientific) for 10 min. Then, the chamber slide was placed on
an onstage incubator equipped with A1-ECLIPSE Ti2-E.
Time-lapse sequence images of microglia were acquired
every 10 s. All images were processed for quantification
using NIS-Elements (Nikon), IMARIS (Bitplane, Zurich,
Switzerland) and ImageJ software programs.
Brain infusion and drug delivery using osmotic pumpin vivo

The mice were anesthetized using a combination of ketamine and isoflurane. Initially, ketamine was administered
via intraperitoneal injection, followed by induction ofanesthesia with isoflurane inhalation. Throughout thesurgical procedure, anesthesia was maintained with continuous administration of isoflurane. A hall for microneedle injection was created in the skull using a dental drill atcoordinated 0.5 mm in the anteroposterior axis, ± 1 mm
in the mediolateral axis and − 2.5 mm in the dorsoventral
axis from bregma [51]. Subsequently, a microneedle (28gauge) from the brain infusion kit (Alzet Osmotic Pump,
CA) was inserted into the intracerebroventricular (ICV)Lee et al. Journal of Neuroinflammation(2024) 21:196

space of the mouse. The osmotic pump (model 1003D,
delivery rate 1.026 μL/hour, duration for 3 days) containing Pyk2-Inh or β-amyloid solution (10 μg/100 μL),
was assembled with the brain infusion kit, and thenimplanted underneath the skin on the mouse’s back. Eachreagent was infused into the osmotic pump to maintaina constant rate of infusion for 3 days. Both Pyk2-Inh andβ-amyloid were diluted in fresh sterile artificial cerebrospinal fluid (ACSF composition: Na 150 mM; K 3.0 mM;
Ca 1.4 mM; Mg 0.8 mM; P 1.0 mM; Ci 155 mM) at 37 ℃
for 10 min before placing on ice. Vehicle group was
injected with only ACSF instead of Pyk2-Inh to matchthe conditions of the experimental treatments. For the
first 3 days, Pyk2-Inh or vehicle was injected into ICV
region through an osmotic pump. This was followed by
β-amyloid injection into each groups, with brain analysis performed 3 days later (Fig. 5A). All evaluations for
microglial (surface, volume, length, phagocytosis etc.),
neuronal cell (neu-N+analysis) parameters were conducted on the final day of the experiments.
Acute brain slice and imaging preparation

Mice were decapitated under anesthesia. The brains weredissociated on cold ACSF and sliced using a brain slicer
at 1 mm thickness [52, 53]. Then, brain slices were fixedwith 4% paraformaldehyde solution for 1 h. For further
immunohistochemistry analysis, brain slices were incubated in the RapiClear 1.52 solution as per the manufacturer’s instructions (SunJin Lab’s, Hsinchu City, Taiwan).
The primary antibody of NeuN (Anti-mouse, Abcam,
ab104224, 1:100) was used for immunohistochemistry.
All slices were analyzed using a laser confocal scanningmicroscope (A1-ECLIPSE Ti2-E, Nikon, Japan) at X60
magnification.
Measurement of IL‑6 by ELISA

The human IL-6 expression levels in the culture supernatant were analyzed using the human IL-6 ELISA ­MAX™
Deluxe set (Biolegend, CA) according to the manufacturer’s instructions. Cells were seeded at a density of 1 × ­104
cells/well in a 96-well plate and cultured for 24 h. Cells
were pretreated with Pyk2 inhibitor as indicated concentration for 3 h and the procedure was followed by exposure to 1 μg/mL LPS (Escherichia coli serotype 0111:B4,
Sigma) for 24 h. The supernatant was collected after centrifugation was stored at − 20 ℃ until further use.
Statistical analysis

Results were shown as mean ± SEM. Statistical significance was determined using GraphPad Prism 9 (GraphPad Software Inc., CA) software program. The data wereanalyzed using Student’s t test for simple comparisonsor One-way ANOVA for multiple comparisons. P-value

Page 5 of 19

of < 0.05 represented statistical significance. NS indicatesthat the result was not significant.

Results
Pharmacological inhibition of Pyk2 activity leads
to the multinucleation of microglia

Pyk2−/− mice are viable and fertile, without disability indevelopment or behavior [54]. However, macrophages
isolated from Pyk2−/− mice were impaired to migrate in
response to chemokine stimulation [54]. To investigate
the morphological and functional effects of Pyk2 inhibition in microglia, MG6 cells were treated with various
doses (range 100 to 1000 nM) of PF-431396 (hereafterreferred to as Pyk2-Inh). Morphological analysis revealed
the presence of multinucleated microglia in MG6 cells
treated with 1000 nM Pyk2-Inh for 24 h whereas suchcells were absent in the vehicle-treated group (Fig. 1A).
Multinucleated microglia treated with Pyk2-Inh also
showed intracellular expression of ionized calciumbinding adaptor molecule 1 (Iba-1, green), a characteristic marker of activated microglia, as well as Tubulin(magenta), a constituent of the cellular cytoskeleton(Fig. 1A). Significantly, quantitative morphometric analysis showed that more than 30% of microglia were multinucleated when treated with 1000 nM Pyk2-Inh for 24 h(Fig. 1B and C).
Next, we evaluated the effects of Pyk2 inhibition on
MG6 cell proliferation using the Cell Counting Kit-8(CCK-8). Colony-Stimulating Factor 1 (CSF1) is known
to regulate the survival, activation and proliferation of
microglia [55], which are the resident immune cells of
the CNS. By treating microglia with CSF1 showed tosignificantly enhance the cell proliferation, doublingcompared to untreated conditions (Fig. 1D). Our findings revealed a dose-dependent response to Pyk2-Inhtreatment for 24 h. Specifically, MG6 cell proliferation
remained unchanged at concentrations up to 100 nM
Pyk2-Inh (97.46 ± 3.11%) compared to the CSF1-treatedcontrol group (100 ± 1.98%). However, a significant
decrease in proliferation was noted in cells treatedwith 500 nM (96.60 ± 2.24%) and 1000 nM concentrations (96.50 ± 1.75%) (Fig. 1D). Microglial survival
relies on the CSF1 and CSF1 Receptor (CSF1R) signaling pathway, which includes Pyk2, Fak, Src, and Erk(Fig. 1E and F). Phosphorylated Pyk2 levels increased
in response to CSF1 treatment for 10 min but weresignificantly reduced following Pyk2-Inh treatment,
which indicates Pyk2 activity was effectively inhibited, with the p-Pyk2/Pyk2 ratio decreasing from1.35 ± 0.08 to 0.26 ± 0.11-fold. Similarly, Pyk2-Inh treatment led to decreased phosphorylation of Fak and Src,
with fold changes of 1.48 ± 0.17 to 0.22 ± 0.06-fold and1.38 ± 0.07 to 0.24 ± 0.11-fold, respectively, comparedLee et al. Journal of Neuroinflammation

A

Control(2024) 21:196

Page 6 of 19

B

Pyk2-Inh (1000 nM)

10 μM

Phase contrast

10 μM

DAPI
An-Iba1 An-β-Tubulin DAPID
Proliferation (control %)Mulnucleated cell, no. nuclear ≥2

E

*

150

*
n.s

****
100

50

0
CSF1
Pyk2-Inh

0

+
0

+
100

C

Pyk2-Inh (1000 nM)

+
500

CSF1
Pyk2-Inh

−
−

+
−

+
+

CSF1
Pyk2-Inh

−
−

+
−

+
+

p-Pyk2116 kDa

p-Erk

42/44 kDa

Pyk2

116 kDa

Erk

42/44 kDa

p-Fak

125 kDa

p-Src

60 kDaFak

125 kDa

Src

60 kDaMannoseReceptor

175 kDa

Tubulin

43 kDa+1000 (nM)F

Fig. 1 Inhibition of Pyk2 signaling induces multinucleation formation in microglia. A Morphological changes between mononuclear
and multinucleated microglia. Immunocytochemistry of MG6 cells stained with anti-Iba1 (green) and anti-β-tubulin (magenta). Nuclear DNA
was labeled with DAPI (white). Scale bar is 20 μm. B Cells were treated with Pyk2-Inh for 24 h. Phase contrast images (upper panel) and DAPI stained
images (lower panel) were shown. Circles are indicated as multinucleated cells. C The quantification of the number of multinucleated cells/well(control: n = 4, Pyk2-Inh-500 nM: n = 4, Pyk2-Inh-1000 nM). D Cell proliferation was assessed by CCK-8. MG6 cells were treated with 100 μg/mL CSF1
and indicated concentration of Pyk2-Inh (n = 6 per group, One-way ANOVA, p < 0.05). E Representatives immunoblot images. The ratio of p-Pyk2/
Pyk2, pFAK/FAK, p-ERK/ERK, p-Src/Src and Mannose receptor/Tubulin were described under the blot. Pyk2-Inh was administered as a pre-treatment
two hours before stimulation with CSF1 (100 μg/mL). F Image quantification and analysis were performed. The same experiments were repeatedthree times. All full-length uncropped original western blots are included in a Sup. Fig. 4. Values are mean ± SD. #: negative control (without CSF1) vscontrol (with CSF1), ##: p < 0.01; *: control (with CSF1) vs Pyk2-Inh (with CSF1), *: p < 0.05; **: p < 0.01; ***: p-value < 0.001; ****: p-value < 0.0001

to untreated conditions. Meanwhile, the expression of
p-Erk signaling notably increased upon treatment with
the Pyk2-Inh, with the p-Erk/Erk ratio increasing from0.82 ± 0.17 to 1.54 ± 0.14-fold. The mannose receptor,

known to be expressed in microglia, could participate
in multiple physiologic and pathologic conditions [56].
However, treatment with Pyk2-Inh did not elicit any
significant changes.

Lee et al. Journal of Neuroinflammation(2024) 21:196

Page 7 of 19A

Pyk2-Inh (1000 nM)

ControlB

00’75”

04’30”

06’30”

06’55”

08’35”

14’60”

20’10”

22’60”25 μM

ControlC

Pyk2-Inh (1000 nM)

25 μM

25 μM

An-Pericentrin An-Tubulin Phalloidin DAPI

Fig. 2 Multinucleated cells are generated from abscission failure in cytokinesis, not cell fusion. A Dynamics of cell division by time-lapse video
microscopy. The phase contrast images of the microglia’s mitosis at different times. Nuclei were observed with Hoechst 33,342 staining. Arrowheads
indicate representative images from anaphase to cytokinesis (control: n = 4, Pyk2-Inh: n = 4). B Cell division was quantified, categorizing it
as either appropriate or inappropriate, in both control (n = 4) and Pyk2-Inh treated groups (n = 4). C Fluorescence images of microglia cells showing
the stages of cell division. Immunocytochemistry of MG6 cells stained with anti-Pericentrin (red), anti-β-tubulin (green) and phalloidin (white).
Nuclear DNA was labeled with DAPI (blue). Independent experiments were performed at least three times

Abnormal abscission during cytokinesis, rather than cellfusion, leads to multinucleation in microglia treatedwith Pyk2‑Inh

To better understand the process of multinucleation,
live cell imaging was conducted after treatment with
Pyk2-Inh (Fig. 2A). In the control group, microglial
cell membranes constricted successfully during telophase, with the separated chromosomes (stained blue
with Hoechst 33,342). Following cell division, thesecells naturally divided into two distinct daughter cells(Fig. 2A upper panel and Sup. Movie 1). In contrast,
microglia treated with 1000 nM Pyk2-Inh showed
divided chromosomes aligning at the centromere, withround shape of mature nuclei forming (confirmed with
Hoechst 33,342 staining). Despite cell membrane contraction, these cells ultimately regressed to a single

entity while retaining two nuclei over time (Fig. 2A
lower panel and Sup. Movie 2). In Fig. 2B, the abnormalities in cell division were quantified for the control
group (n = 4) and the Pyk2-Inh-treated group (n = 4).
The percentage of appropriate and inappropriate divisions was compared to the total number of cells ineach treatment group. As a result, the control group
showed appropriate division (14.3 ± 7.69%) and inappropriate division (0%), but the Pyk2-Inh-treated group
showed appropriate division (2.42 ± 1.84%) and inappropriate division (10.76 ± 4.78%). Statistically, within
the Pyk2-Inh-treated group, the comparison between
appropriate and inappropriate division showed a significant increase in inappropriate divisions. Additionally,
compared to the control group, the Pyk2-Inh-treatedLee et al. Journal of Neuroinflammation(2024) 21:196

group showed a significant decrease in appropriate
divisions and a significant increase in inappropriate
divisions. This demonstrates that Pyk2 inhibitor treatment induces multinucleation due to abnormalities indivision.
To investigate the cell division process more closely,
immunofluorescence staining was utilized to highlight actin, microtubules, and centrosomes (Fig. 2C).
This approach revealed that in multinucleated microglia treated with Pyk2-Inh, microtubule bundle (shownin green, indicated by red arrow) continued to persist
between two cells after cell division and eventually toreaggregate into a single entity (Fig. 2C lower panels).
The cleavage furrow in this process may be functionallydefected, which is a crucial player in constricting thecell membrane and separating it into two daughter cells[57]. Since centrosomes are pivotal during cell division[58, 59], we confirmed whether appropriate cell divisionoccurs by constructing an appropriate division apparatus around each nucleus in multinucleated microglia by
Pyk2-Inh. There were no observed defects in centrosome
formation, as indicated by staining with anti-Pericentrin(shown in red). These results indicate that multinucleation induced by Pyk2 inhibition results in improper cell
division due to abscission failure attributed to cytoskeletal abnormalities, rather than cell fusion.
Multinucleated microglia enhance phagocytotic function
compared to mononucleated microglia

To assess functional differences in phagocytic activity between multinucleated and mononuclear microglia
after Pyk2 inhibition, phagocytic function was quantified. Both mononuclear and multinucleated microglia,
isolated by FAC sorting following Pyk2-Inh treatment(Sup. Figure 1), were then exposed to pHrodo BioParticles-Red, a fluorogenic phagocytic substrate derived from
E. coli. Microglia treated with Pyk2-Inh and untreated(control) mononuclear microglia efficiently engulfed the
BioParticles (Fig. 3A upper panel). To further examine
the differences in phagocytosis between mononucleatedand multinucleated microglia, cells treated with Pyk2-Inh

Page 8 of 19

underwent confocal microscopy analysis to produce
three-dimensional images of the phagocytosed BioParticles (Red). For imaging analysis, the entire cell cytoskeletal was stained with phalloidin (green), and the nucleus
was stained with DAPI (white) to distinguish between
mononuclear and multinuclear cell (Fig. 3A upper panels). Subsequently, these images were quantified using
IMARIS imaging software (Fig. 3A lower panel and B).
We observed no difference in phagocytosis between
control and Pyk2-Inh treated mononucleated microglia;
however, Pyk2-Inh treated multinucleated microglia
exhibited a significant increase in phagocytosis (Fig. 3B).
The average number of BioParticles phagocytosed percell was counted as 86.46 ± 38.52 in the naïve control
group (n = 4 per group), 106.7 ± 69.63 in the Pyk2-Inhmono group (n = 4 per group), and 199.05 ± 98.91 in the
Pyk2-Inh-multi group (n = 4 per group). Each independent experiment was conducted at three times to analyze
the number of cells in Fig. 3B.
Next, we examined how phagocytosis evolved overtime following Pyk2-Inh treatment. Cells separated
through FAC sorting were cultured, and exposed to
pHrodo BioParticles, and the fluorescence intensity wasrecorded at designated time intervals (Fig. 3C and D).
Notably, in multinucleated cells, a marked enhancementin fluorescence, indicative of increased phagocytic activity, was observed beginning 3 h after pHrodo BioParticles treatment (Fig. 3D). Compared to the control group(phagocytosis average 100%), the phagocytosis of Pyk2Inh treated multinucleated microglia increased morethan twofold to 226.21% at 4 h (Fig. 3E). In contrast,
the Pyk2-Inh treated mononucleated microglia group
showed a decreased, averaging 81.77%, although this was
not statistically significant. Additionally, considering thatmultinucleated cells may either be short-lived or revert
to a mononuclear state via cytokinesis, we investigated
the survival differences between multinucleated andmononucleated microglia. The survival rates of each cell
type, post-sorting, were monitored over 24 h using theCCK-8 (Fig. 3F). Multinucleated cells by Pyk2-Inh demonstrated a slight increase in cell proliferation compared(See figure on next page.)
Fig. 3 Comparison of phagocytotic function in multinucleated versus mononucleated microglia. A Immunofluorescence imaging depicting
phagocytosis of pHrodo Red E.coli BioParticles by mononuclear and multinucleated MG6 cells after exposure to Pyk2-Inh. Cells were treatedwith pHrodo Red E. coli BioParticles for 1 h and fixed with paraformaldehyde. Actin cytoskeleton and nuclear DNA were stained with phalloidin(green) and DAPI (white), respectively. Engulfed red fluorescein were detected by microscopy and analyzed by IMARIS software (lower panel). B
Bioparticles engulfed in each cell were quantified and compared. multi/n means divided by the number of nuclei in multinucleated microglia (n = 4
per group). C Mononuclear and multinucleated microglia were separated by fluorescence-activated cell sorting (FACS) and treated pHrodo-Red
E. coli for 30 min. D, E Sorted Cells were labeled with pHrodo Red E. coli BioParticles for the indicated time and fluorescein was measured
by fluorescence detector (n = 8 per group). On 4-h incubation with BioParticles, fluorescein intensity was indicated in bar graph. F CCK-8 assay
was used to measure the proliferation of each type of microglia. Sorted cells were incubated for 24 h and proliferation assay were conducted (n = 4
per group). Values are mean ± SD. *: p < 0.05; **: p < 0.01; ***: p-value < 0.001; ****: p-value < 0.0001

Lee et al. Journal of Neuroinflammation(2024) 21:196

Page 9 of 19

Pyk2-Inh (1000 nM)

A

ControlB

Mulnuclear

Mononuclear***

pHrodo BioParcle-Red

Phalloidin

Number of Spots in cell

800

DAPI

*
n.s

600
400
200
0

mono mono multi/n multiCon

Pyk2-Inh (1000 nM)

IMARISCD

Pyk2-Inh (1000 nM)
Control

Mononuclear

Mulnuclear

Phase contrast

10 μM

pHrodo

10 μM

Phagocytosis

At 4 hour

E

F

***
n.s

**

4 Hour

300
200
100
0

Con

Mono

*

150
Proliferation (control %)400

Aer 24 hours

Multi

Pyk2-Inh (1000 nM)

n.s

*

100

50

0

Con

Mono

Multi

Pyk2-Inh (1000 nM)

Fig. 3 (See legend on previous page.)to the naïve control or Pyk2-Inh-mononuclear microglia, assuming that their cell size is larger than that ofmononuclear cells. In contrast, mononucleated microglia
treated with Pyk2-Inh showed no significant proliferation
alteration.

Lee et al. Journal of Neuroinflammation(2024) 21:196

Microglia treated with Pyk2‑Inh demonstrate normal
lysosomal activity in response to soluble Aβ species

An increase in phagocytic activity was observed following Pyk2-Inh treatment, particularly in multinucleatedmicroglia. Subsequently, we assessed whether phagocytosed soluble Aβ species could be effectively degradedby measuring lysosomal activity. Microglia treated with
Pyk2-Inh were additionally exposed to an AD-relevant
substrate, Aβ peptides (1–42, 500 ng/mL). After 24 h,both control and Pyk2-Inh treated microglia cells were
harvested, lysed, and subjected to immunoblotting tomeasure the relative protein levels of phosphorylated
Pyk2, Lamp1, and phosphorylated Erk (Fig. 4A and
B). Exposure to Aβ significantly increased the phosphorylation of Pyk2 (ratio 1.34 ± 0.29 to 4.43 ± 1.25fold) and reduced Lamp1 expression (ratio 2.69 ± 0.57
to 1.59 ± 0.37-fold) under CSF1 presence, indicating a
suppression of lysosomal activity in microglia (Fig. 4A
and B). Interestingly, Pyk2 phosphorylation was suppressed by Pyk2-Inh treatment (ratio 4.43 ± 1.25 to
0.45 ± 0.11-fold), and Lamp1 expression levels were significantly restored following treatment with Pyk2-Inh(ratio 1.59 ± 0.37 to 2.92 ± 0.54-fold). Similar to Fig. 1E,
the phosphorylation of Erk increased with or without
Aβ peptides under Pyk2-Inh treatment. To visualize
amyloid phagocytosis, microglia cells were treated with
HiLyte-555 labelled Aβ42 (500 ng/mL). It was observedthat microglia treated with Pyk2-Inh exhibited enhanced
phagocytic activity and higher affinity for amyloid peptide. Quantitative analysis showed that the Pyk2-Inh(1000 nM) treated group engulfed more Aβ42 spots(average of 16.2 per cell) compared to the control group(average of 5.7 per cell) (Fig. 4C and D). Additionally, toconfirm lysosomal activity, we performed a fluorescencedequenching assay using D­ Q™ Green BSA to assess protease function (Fig. 4E and F). After administered with
DQ green BSA for 3 h, intracellular accumulation of fluorescein derivatives (green) was observed (Fig. 4E). In the

Page 10 of 19

group treated with Pyk2-Inh (1000 nM), fluorescenceintensity of dequenched BODIPY significantly increased
compared to control group, indicating higher lysosomal
activity (Fig. 4F).
The sorted cells were further analyzed for the transcription levels of phagocytosis-related receptors, such as
Trem2, Cd36 and Cd74, along with the phagocytosis substrate gene, Apoe (Fig. 4G). In multinucleated microglia
treated with Pyk2-Inh, Trem2 and Cd36 exhibited significant up-regulation, with 1.9-fold and 3.1-fold increases
in transcriptional levels, respectively, whereas Cd74 andApoe were down-regulated. The transcript level of CD33,
a gene associated with LOAD risk and known to inhibits Aβ phagocytosis, was reduced in Pyk2-Inh-mono but
comparable to Pyk2-Inh-multinucleated cells when compared to control group. Collectively, our findings indicatethat Pyk2 inhibition not only amplifies phagocytosis in
microglia—particularly in multinucleated ones—but alsoenhances the lysosomal activity and the degradation ofphagocytosed substrates within these cells.
Morphological changes in microglia observed in the brainsof Iba‑1 EGFP Tg mice

To examine the in vivo microglial response to Pyk2-Inh,
we established an acute Aβ accumulation model using
Iba1-EGFP Tg mice as illustrated in Fig. 5A. Pyk2-Inhand HiLyte-555 labelled Aβ42 were delivered into themouse brain through a brain infusion kit connected
to an osmotic pump. After 72 h of infusion, the Pyk2Inh treated group exhibited distinct morphologicalchanges—enlarged cell bodies and shortened ramification—compared to typical naïve microglia, which havethin and elongated ramification (Fig. 5B). Morphological changes were quantitatively analyzed using IMARIS
software. In the Pyk2-Inh treated group compared tothe control group, cell surface area (μm2) significantly
increased from 103.322 to 267.263, while ramification
volume (μm3) and length (μm) decreased from 2.11 to(See figure on next page.)
Fig. 4 Pyk2 inhibition stimulates β-amyloid-induced phagocytotic function in multinucleated microglia. A Immunoblot of microglia cells
after treatment with 1000 nM Pyk2-Inh and 500 ng/mL β-amyloid oligomers (1–42). Cells were treated with 100 ng/mL CSF-1 and 1000 nM
Pyk2-Inh for 24 h. Cell lysates were subjected to immunoblotting of p-Pyk2/Pyk2, Lamp1 and p-Erk/Erk. Tubulin was used as a loading control. B
The quantification of immunoblotting data measured by ImageJ. The same experiments were repeated three times. All full-length uncropped
original western blots are included in a Sup. Fig. 5. #: significance between negative control (without CSF1) and control (with CSF1) under β-amyloid
absence, #: p < 0.05; ##: p < 0.01; *: significance between control (with CSF1) and Pyk2-Inh (with CSF1) under β-amyloid presence, *: p < 0.05; **:p < 0.01; ns; not significant. C Immunocytochemical imaging depicted phagocytosis of Aβ (1–42, 500 ng/mL)-Fluor 555 (red) labeled oligomers
by microglia cells after exposure to Pyk2-Inh. Cells were stained with anti-Iba1 (cyan) and nuclear DNA was stained with DAPI (white). Scalebar is 20 μm. D The quantification of number of spots per cell. After obtaining three-dimensional images, β-amyloid are quantified by a spot analysis
using ImageJ. E Lysosomal function was assessed using ­DQ™ Green BSA. Intracellular accumulation of fluorescein derivatives (green) was observed.
Scale bar is 100 μm. F Fluorescence intensities of dequenched BODIPY resulting from proteolysis activity were measured using a fluorescence plate
reader. G The transcriptional expression of Trem2, Cd36, Cd74 and Apoe in control and Pyk2 inhibitor treated group in microglia cells. Values aremean ± SD. *: p < 0.05; **: p < 0.01; ***: p-value < 0.001; ****: p-value < 0.0001

Lee et al. Journal of Neuroinflammation

A(2024) 21:196

B

β-Amyloid (500 ng/mL)

CSF1
Pyk2-Inh

−
−

+
−

+
+

−
−

+
−

+
+

p-Pyk2116 kDa

Pyk2

116 kDa

Lamp1

120 kDa

p-Erk

42/44 kDa

Erk

42/44 kDa

Tubulin

43 kDaC

Page 11 of 19

ControlD

Pyk2-Inh (1000 nM)

An-Iba1 β-amyloid (1-42)-555 DAPI

ControlF

Pyk2-Inh (1000 nM)

DQ Green BSA (de-quenched BODIPY)
Fluorescence Intensity (A.U)

E

100 μM

100 μM

Lysosomal function
11600

ns

11400
11200
11000
10800

10600
Pyk2-Inh

500 1000 (nM)0

DQ Green BSA DAPI

1.5
1.0
0.5
0.0

Con Mono Multi

P=0.0003

1.0

0.5

0.0

Con Mono Multi

Pyk2-InhFig. 4 (See legend on previous page.)Pyk2-Inh

P=0.001
P=0.013

3

2

1

0

Con Mono Multi
Pyk2-Inh1.5

P=0.001

1.0

0.5

0.0

Cd33

P=0.007

Con Mono Multi
Pyk2-Inh

1.5

Cd33 / Gapdh expression(2ΔCT fold change )

n.s

Apoe

Cd36
4

P=0.0005

Cd36 / Gapdh expression(2ΔCT fold change )

2.0

Cd74
1.5

p=0.006

Cd74 / Gapdh expression(2ΔCT fold change )Trem2 / Gapdh expression(2ΔCT fold change )2.5

Apoe / Gapdh expression(2ΔCT fold change )

Trem2

G

n.s
P=0.0103

1.0

0.5

0.0

Con Mono Multi
Pyk2-InhLee et al. Journal of Neuroinflammation(2024) 21:196

Page 12 of 19

A

B

ControlC

Pyk2-Inh

Control

Pyk2-Inh

IMARISIMARIS

20 μM

D

Iba1-GFP β-amyloid

E

5 μM

ControlF

Pyk2-Inh

5 μM

IMARISG

H

Pyk2-Inh

20 μM
Iba1-GFP β-amyloid An-NeuN DAPI

Fig. 5 Altered morphological features and phagocytic function in microglia of Iba-1 EGFP Tg mice brain after Pyk2-Inh infusion. A The
experimental schedule for brain infusion assay in Iba1-EGFP Tg 7-week-old mice (n = 3/group). B Confocal images of GFP positive microglia (green)and β-amyloid (red). Obtained brain slices (thickness ≤ 1.0 mm) were treated with RapiClear 1.52 for tissue clearing and immunohistochemical
assay was conducted. C, D Morphological changes were analyzed by IMARIS software. Cell surface, dendrite volume, and dendrite length were
quantified. E Spot analysis by IMARIS software. F, G Quantification of the number of β-amyloid spots per cell and number of microglia deposits. H
Representative confocal image of multinucleated microglia (indicated red arrow). Brain slices were stained with anti-NeuN (white) and nuclear DNA
was stained with DAPI (white). Values are mean ± SD. *: p < 0.05; **: p < 0.01; ***: p-value < 0.001; ****: p-value < 0.0001

1.78 and from 7.43 to 6.22, respectively (Fig. 5C and D).
Surprisingly, compared to controls, Pyk2-Inh-treatedmicroglia showed increased association with amyloid
spots (Fig. 5E, marked by orange spots). The quantification of yellow fluorescence, which represents the mergeof green and red signal and serves an indicative forinternalized Aβ42, revealed a significantly higher densityof Aβ spots per cell in the Pyk2-Inh-treated group compared to the control group (6.99 for Pyk2-Inh-treated vs.
2.27 for control). Additionally, there was an increased
presence of microglia surrounding the Aβ deposits in the
Pyk2-Inh-treated group compared to the control groupLee et al. Journal of Neuroinflammation(2024) 21:196(9 for Pyk2-Inh-treated vs. 3 for control) (Fig. 5F and G).
We also occasionally observed the multinucleated microglia in the brain tissue (Fig. 5H). These were quantified
as ratio of multinucleated microglia per mononuclear
microglia from each brain section (Sup. Figure 2A and
B). We identified microglia as Iba1-EGFP-positive cells
in brain sections, and defied multinucleated microglia
as those merged with DAPI-positive staining. Quantification was performed by assessing the proportionof multinucleated microglia among the total microglia
count in the imaging. The results showed that the total
microglia count was 42 ± 6.67 in the control group and44.2 ± 7.46 in the Pyk2-Inh treated group, with proportion of multinucleated microglia being 0.017 ± 0.016
in the control group and 0-0.08 ± 0.019 in the Pyk2-Inhtreated group. This indicates a 4.56-fold increase in multinucleated microglia observed in the brain tissue due to
Py2-Inh treatment compared to the control. In addition,
we performed the immunohistochemistry for NeuN,
which is specific marker for neurons. The total count of
NeuN-positive cells was quantified (Sup. Figure 2C and
D). Although Py2-Inh induced multinucleated microglia
in mouse brain tissue, the number of neurons was comparable to the control group. This suggests that Pyk2
inhibition could be an effective approach to improve Aβ
clearance by enhancing microglial phagocytosis and lysosomal functions without being detrimental to neurons.
Pyk2 inhibition attenuates LPS‑induced inflammatory
responses of microglia

Given the varying translatability of murine AD data to
the actual human disease, we proceeded to investigate
the human microglia cell line, HMC3. Initially, we examined whether a Pyk2 inhibitor induces multinucleationin human microglia. As depicted in Sup. Figure 3A, weobserved a significant increase in microglia with two or
more nuclei, with percentages of 14.47% at 500 nM and23.27% at 1000 nM of the Pyk2-Inh, compared to 7.43%
in the control group (Sup. Figure 3B). Activated microglia
play a complex role in neurodegenerative diseases, acting
as both beneficial immune responders and detrimental
inflammatory mediators. Next, we examined the inflammatory response using HMC3 cells. We pre-treatedhuman microglia cells (HMC3) with Pyk2-Inh for 3 h,followed by LPS stimulation for another 3 h. LPS is frequently used as a stimulus to investigate microglial reactivity and pro-inflammatory responses [60–62]. We thenmeasured their inflammatory responses. Pyk2-Inh treatment significantly reduced the mRNA expression levelsof IL1B, IL6, and TNF, well-characterized inflammatory
response genes (Fig. 6A). To corroborate these findings,
secreted IL-6 protein levels in the culture medium, measured by ELISA (Fig. 6B), were significantly lower in the

Page 13 of 19

Pyk2-Inh-pretreated group compared to the LPS-stimulated control group, consistent with the mRNA level
data, further supporting the anti-inflammatory effects of
Pyk2 inhibition. Next, we investigated the downstream
signaling of LPS stimulation using Western blot analysis (Fig. 6C). The inflammatory mater regulator, NF-kB,
is activated by LPS. The phosphorylation of NF-kB rapidly responds to LPS, initiating inflammatory responses.
The expression of NF-kB markedly increased 15 minafter LPS priming compared to 0 min (from 0.51 ± 0.31
to 1.53 ± 0.35-fold). Conversely, in the presence of Pyk2Inh, stimulation of microglia by LPS results in decreased
expression of NF-kB as early as the 0–5-min time point,
significantly decreasing from 1.53 ± 0.35 to 0.92 ± 0.37 at
15 min (Fig. 6D). Thus, the suppression of phosphorylated form of NF-kB due to Pyk2-Inh suggests a reductionin LPS-induced inflammatory responses mediated by
NF-kB inhibition.

Discussions
Effective clearance of Aβ oligomers and insoluble fibrils
by microglia is important for preventing or slowing down
senile plaque formation and other pathophysiologicalchanges in AD. During the preclinical and early stages ofAD, activating microglia is crucial for efficient Aβ clearance [63]. Enhanced phagocytosis improves the clearanceof pathogens, dead cells, and debris, which is essential for
preventing infections and maintaining a healthy neuralenvironment [64]. In contrast, late-stage dysfunction andpersistent activation of microglia and enhanced phagocytosis can lead to chronic inflammation. In some cases,
overactive phagocytosis can result in the engulfment anddestruction of healthy neurons and synapses, contributing to neurodegeneration and cognitive decline [65]. This
complex, dual nature of microglia in AD presents challenges in pinpointing targets and demonstrating treatment effectiveness. Therefore, understanding the balance
and regulation of microglia phagocytosis is crucial fordeveloping therapeutic strategies for a variety of neurological conditions.
The pathogenesis of AD is multifactorial, involving
several key pathological hallmarks: amyloid plaques
accumulation, tau protein abnormalities and neuroinflammation [66]. Amyloid plaques are composed of the
β-amyloid peptide Aβ1-42, a toxic derivative of the amyloid precursor protein (APP), while neurofibrillary tangles are formed from aggregates of hyperphosphorylatedTau, a microtubule-binding protein. Recent GWAS haveidentified PTK2B as a susceptibility locus for AD [29–31, 67–69], pointing to the Pyk2 may play a role in the
AD pathology. Pyk2 phosphorylation shows heightened
activity in wild-type mouse brain slices exposed to Aβoligomer and in aged APPswe/PS1ΔE9 transgenic mouseLee et al. Journal of Neuroinflammation

IL1B
4

2.0

Relative expression / RPLP0

Relative expression / RPLP0

IL6

####

2.5

*

1.5

**

1.0

****

0.5

0.0
Pyk2-Inh

0

0

0.5

1

8

*

3

**
2

**

1

0
Pyk2-Inh

5 (µM)

0

0

0.5

5

15 30

60

5 (µM)

0

5

15 30

60

6

*
**

4

**
2

0
Pyk2-Inh

0

0

0.5

1

5 (µM)

LPS(1 µg/mL)D

Pyk2-Inh (5 μM)
01

LPS(1 µg/mL)

C

B

TNF

###

####

LPS(1 µg/mL)

LPSPage 14 of 19

Relative expression / RPLP0

A(2024) 21:196

min

p-NF- B

65 kDaNF- B

65 kDaβ-acn42 kDaFig. 6 Pyk2 inhibition reduces LPS-induced inflammation in human microglia. A The transcriptional expression of IL1β, IL6 and TNF in control
and Pyk2-Inh treated group in human microglia cells, HMC3. B The secreted protein level of IL-6 was measured by ELISA. C The time-course
Immunoblotting images of phosphor-NF-kB and NF-kB in HMC3 cells after LPS stimulation and with or without Pyk2-Inh. β-actin was used
for loading control. The same experiments were repeated three times. D Quantification of immunoblotting data was measured by ImageJ.
All full-length uncropped original western blots are included in a Sup. Fig. 6. Values are mean ± SD. #: significance between negative control
and LPS control, #: p < 0.05; ##: p < 0.01; *: significance between LPS control and Pyk2-Inh treated groups, *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns:
not significant

brain [70, 71]. Pyk2 is part of a signaling cascade triggered by oligomeric Aβ (Aβo) [72]. This cascade involves
the cellular prion protein (PrPc), which act as a receptorof Aβo, and interacts with mGluR5, Homer1b/c, Pyk2,
and ­Ca2+/calmodulin-dependent protein kinase II [70].
These cascade phosphorylated Tau, potentially facilitating its aggregation and the formation of neurofibrillary tangles. The absence of Pyk2 exacerbates mutant
Tau-dependent phenotypes in PS19 mice, partly due toincreased LKB1 and MAPK activity [73]. These data suggest that while Pyk2 activity mediates Aβ-driven deficits
AD, it also suppresses Tau-related phenotypes. Additionally, Pyk2 KO also improves dendritic spine loss induced
by Aβo, likely by alleviating the inhibition of Graf1c, a
RhoA GTPase-activating protein by Pyk2 [74]. Similarly,
our results showed that despite the increased phagocytic
function of microglia by Pyk2 inhibition, there was no
significant difference in the number of neurons measured by the NeuN marker between the control group andPyk2-Inh treated group. Therefore, Pyk2 inhibition may
does not affect neuronal loss. Collectively, these findings indicate that Pyk2 may contribute to tauopathy bydirectly and indirectly modifying Tau phosphorylation
at multiple sites. Thus, Pyk2 is a promising candidate forlinking Aβ and tau pathology within the amyloid cascade
hypothesis.
In the current study, we developed an assay to assess
microglial function, exploring Pyk2-Inh’s effects on Aβ
clearance. Notably, Pyk2 inhibition induced multinucleated microglia formation, associated with improved
phagocytic and proteolytic capabilities. Multinucleated microglia represent a unique and intriguing aspectof microglial biology, frequently associated with severe
chronic pathological conditions [75]. These cells areprominently observed in conditions such as HIV/AIDS
encephalopathy [75, 76], which suggests their genesis may be attributed to cell fusion processes activatedby viral presence within microglia. The formation ofLee et al. Journal of Neuroinflammation(2024) 21:196

multinucleated cells deviates from the conventional
behavior of microglia, which are typically characterized by a territorial disposition and exhibit pronounced
contact inhibition, delineated by a slim and extensively
branched morphology [77]. The precise biological purpose and consequence of multinucleated microglia formation are yet to be elucidated. These cells may arise
through mechanisms such as failed cell division, cellfusion or other forms of cellular stress [76, 78, 79]. Unlike
osteoclasts, where multinucleation is irreversible [80],
microglial multinucleation is reversible. This raises questions about the functional relevance of this phenomenon.
The increased size of multinucleated microglia hints at
a potential specialization for enhanced phagocytosis,
allowing them to engulf larger cellular debris or particulate matter.
Pyk2 also plays an important role in inflammatory
diseases [54, 81]. In vitro, Pyk2 acts downstream of
PKC to mediate the secretion of neurotoxic factors by
microglia in response to Aβ stimulation [82]. TRPM2, a
ROS-sensitive calcium channel expressed in neurons,
astrocytes, and microglia, has been linked to numerous
neurological diseases [83]. In monocytes (circulating
macrophages), TRPM2 activates the Pyk2/ERK pathway,
leading to chemokine production and increased inflammation [84]. This pathway is crucial for microglia activation by Aβ1-42, resulting in the release of cytotoxic tumor
necrosis factor-ɑ (TNF-ɑ). Pyk2 enhances this process
through positive feedback on the TRPM2 channel [85].
In microglia treated with Pyk2-Inh, we observed a significant decrease in LPS-induced inflammatory response,
accompanied by decrease of phosphorylated NF-kB andreduced transcriptional expression of inflammatory
genes, including IL1β, IL6, and TNF. This reduction in
inflammatory response could be attributed to Pyk2 inhibition potentially interfering with the Pyk2 and MyD88
interaction [42]. MyD88 is essential for linking LPS-activated TLRs with the Pyk2/IRF4 complex [42, 86], underscoring the significance of targeting these interactions intherapeutic strategies designed to enhance Aβ clearance
mechanisms in neuroinflammatory and neurodegenerative disorders while simultaneously reducing pro-inflammatory signals.
One limitation of this study is the specificity of the Pyk2
inhibitor, PF-431396. This small molecule inhibitor targets PTK2B, but due to Pyk2’s 65% homology and shareddomain structure with FAK [87–89], PF-431396 canalso inhibit FAK activity, as demonstrated in immunoblot assay (Fig. 1E). Despite this limitation, our research
identifies a significant benefit of PF-431396 in triggering multinucleated microglia formation, associated withenhanced phagocytic capabilities. The exclusive enhancement of phagocytic activity in multinucleated microglia

Page 15 of 19

treated with PF-431396 underscores the potential significance of Pyk2/FAK pathways in microglial phagocytosis, meriting further exploration. Additionally, ongoing
Phase II clinical trials of FAK inhibitors for breast cancer
metastasis inhibition [90], and studies on the efficacy of
dual Pyk2/FAK inhibitors in such contexts, underscorethe broader therapeutic potential of targeting these signaling pathways [91].
There is a critical need for further research to elucidate the mechanisms that Pyk2 plays in diverse AD animal models and to assess their applicability to humandiseases. AD genetic mouse models (e.g., App/Psen1 or
5xFAD) are commonly used to mimic the pathophysiology of AD and evaluate potential therapeutic interventions [92, 93]. However, the fact that we only validated
the effects of the Pyk2 inhibitor in an acute AD modeland did not assess its efficacy in an AD genetic mousemodel raises important consideration for discussion.
While acute AD models allow for rapid assessment oftherapeutic effects, AD genetic mouse model moreclosely recapitulate the progressive nature and complexity of AD observed in humans. Therefore, the lack of validation in AD genetic models raises questions about thetranslational potential of our findings and underscores
the need for further investigation in models that better represent the disease phenotype. Lastly, considering
alternative models or experimental approaches that better reflect the genetic complexity and progressive natureof AD can enhance the translational relevance of ourfindings. This may include utilizing transgenic mousemodels expressing familial AD mutations or incorporating in vitro models with human-derived cells and genetic
variants associated with AD.
Supporting the positive role of Pyk2, studies haveshown reduced Tyr402 phosphorylation levels in the hippocampus can be mitigated by overexpressing Pyk2 inthis brain region. This overexpression restores autophosphorylated Pyk2 levels, enhances synaptic markers, andimprove performance in behavioral tasks, despite a slightincrease in plaque number in 5XFAD mice [94]. Furthermore, overexpressing Pyk2 in hippocampal neurons
within a microfluidic culture system has been shown toprotect these neurons from Aβ1-42 toxicity [95]. Although
the precise role of elevated Pyk2 expression in microglia and its contribution to AD pathology has yet to be
explored, it is conceivable that Pyk2 significantly modulates microglial activity, neuroinflammatory responses,
and synaptic integrity. While inhibition of Pyk2 has beenshown to reduce pathological tau phosphorylation andneuroinflammation as discussed in earlier, overexpression studies indicate that Pyk2 may also play a protective role by enhancing synaptic plasticity and neuronalsurvival. The seemingly contradictory effects of Pyk2

Lee et al. Journal of Neuroinflammation(2024) 21:196

inhibition and overexpression could be attributed to thespecific cellular contexts or stages of the disease in which
Pyk2 activity is modulated. Additionally, compensatory
mechanisms and the precise levels of Pyk2 activity might
critically influence the outcomes. Therefore, further studies are required to elucidate the conditions under which
Pyk2 inhibition or overexpression can be therapeuticallybeneficial, take into account the functional diversity andregulatory mechanisms of Pyk2 in AD pathology.
In this study, we found a novel discovery that microglia
undergo multinucleation upon inhibition of Pyk2 using
PF-431396, and we demonstrated that this multinucleation correlates with an increased ability of the cells toengulf Aβ. Specifically, activated microglia are known
to exert phagocytotic activity particularly heightenedby inflammation. However, these multinucleated microglia not only do not exacerbate inflammatory responses
induced by LPS but also facilitate the clearance of Aβ.
Intriguingly, we noted an increased affinity for amyloid spots around Pyk2-Inh-treated microglia, particularly in multinucleated microglia in vitro, and observed
enhanced migration of microglia around amyloid deposits in vivo. These findings suggest a potential therapeutic
avenue for inflammatory neurodegenerative disorders,
where inflammation could be mitigated while promoting
Aβ clearance.
Abbreviations
Aβ	β-Amyloid
PTK2B	Protein Tyrosine Kinase 2 Beta
EGFP	Enhanced Green Fluorescent Protein
LPS	Lipopolysaccharide
CNS	Central nervous systemAD	Alzheimer’s disease
PD	Parkinson’s diseaseMS	Multiple sclerosis
LOAD	Late-onset Alzheimer’s disease
FAK	Focal adhesion kinase
GWAS	Genome-wide association study
ICV	Intracerebroventricular
Tg	Transgenic
ACSF	Artificial cerebrospinal fluid
FACS	Fluorescence-activated cell sorting

Supplementary Information

Page 16 of 19

mononucleated microglia (control=5, Pyk2-Inh=5). C) Representative
images of immunofluorescence staining for anti-NeuN (white). D) The
total count of NeuN-positive cells was quantified (control=7, Pyk2-Inh=7).
Figure 3 Pyk2 inhibition induces multinucleation in human microglia cells.
HMC3 cells were seeded at a density of 1 X ­104 cells per well in a 96-wellplate and incubated with or without Pyk2-Inh at concentrations of 500
and 1000 nM. A) After 24 hours, the cells were fixed with 10% formalinand stained with phalloidin (red) for the cytoskeleton and DAPI (blue)
for nuclei. B) Microglia with two or more nuclei per cell were classified
as multinucleated Yellow arrows indicated the multinucleated microglia.
(control=5, Pyk2-Inh 500 nM=5, Pyk2-Inh-1000 nM=5). The experiments
were repeated three times. Figure 4 All full-length uncropped original
western blots from Fig. 1E. Figure 5. All full-length uncropped original
western blots from Fig. 4A. Figure 6. All full-length uncropped original
western blots from Fig. 6C. Movie 1. Cell division in the control group by
time-lapse video microscopy. Nuclei were stained with Hoechst 33342
staining. Movie 2. Cell division in Pyk2-Inh treated group by time-lapse
video microscopy. Nuclei were stained with Hoechst 33342 staining.
Movie 3. Morphological feature analysis of control microglia by IMARIS.
Movie 4. Morphological feature analysis of Pyk2-Inh treated microglia by
IMARIS. Table 1 Oligonucleotide primer sequence for RT-qPCR.
Author contributions
J-W.L. and S.W.K conceived the study, and wrote the manuscript; J-W.L.
designed the experiments and K.M., H.W., and T.T. performed the experiments.
I-H. L performed the data interpretation. K.H., Y.Y., Y.K., A.H. and T.I. provided
insightful discussion. S.W.K. supervised the project. All authors approved thefinal manuscript.
Funding
This work was supported by Grant-in-Aids for Scientific Research from theJapan Society for the Promotion of Science (JSPS KAKENHI) grant number
23K09116, the Takeda Science Foundation, and the JSBMR Rising Star Grant,
which were awarded to J-W. L. This work was partly supported by the National
Institute of Health (Grand numbers: R01NS129188 and U01TR002623) to S.W.K.
Availability of data and materials
No datasets were generated or analyzed during the current study.

Declarations
Ethics approval and consent to participate
Experiments using mice were approved by the Institutional animal care anduse committee of Hokkaido University, Japan and were performed in accordance with the committee’s guiding principle (approval no.: 21-0018).
Competing interests
The authors declare no competing interests.
Received: 7 June 2024 Accepted: 31 July 2024

The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12974-​024-​03192-7.
Additional file 1: Figure 1 Fluorescence-activated cell sorting (FACS) Gating
strategy. The gating strategy for FACS aimed to isolate the mononucleated cells and multinucleated cells. MG6 cells were stained with Hoechst
33342 for 5 minutes before being loaded into the FACS. Following theexclusion of the cell debris and doublet cells, the remaining cells weresegregated based on size differences, with P4 representing mononucleated cells and P5 representing multinucleated cells. Figure 2 Analysis ofmultinucleated microglia and NeuN-positive cells in mouse brain tissue.
A) Iba1-EGFP positive microglia (green) were visualized and mergedwith DAPI staining (blue). Microglia with two or more nuclei per cellwere classified as multinucleated. Red arrows indicated the multinucleated microglia. B) The data is presented as the ratio of multinucleated toReferences
1. McKhann GM, Knopman DS, Chertkow H, Hyman BT, Jack CR Jr, Kawas
CH, Klunk WE, Koroshetz WJ, Manly JJ, Mayeux R, et al. The diagnosis
of dementia due to Alzheimer’s disease: recommendations from thenational institute on aging-Alzheimer’s association workgroups on
diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement.
2011;7:263–9.
2. Braak H, Braak E. Neuropathological stageing of Alzheimer-relatedchanges. Acta Neuropathol. 1991;82:239–59.
3. Hur JY. gamma-Secretase in Alzheimer’s disease. Exp Mol Med.
2022;54:433–46.

Lee et al. Journal of Neuroinflammation

4.

(2024) 21:196

Bertram L, Tanzi RE. Thirty years of Alzheimer’s disease genetics:
the implications of systematic meta-analyses. Nat Rev Neurosci.
2008;9:768–78.
5. Coric V, van Dyck CH, Salloway S, Andreasen N, Brody M, Richter RW,
Soininen H, Thein S, Shiovitz T, Pilcher G, et al. Safety and tolerability of
the gamma-secretase inhibitor avagacestat in a phase 2 study of mild
to moderate Alzheimer disease. Arch Neurol. 2012;69:1430–40.
6. Egan MF, Kost J, Tariot PN, Aisen PS, Cummings JL, Vellas B, Sur C, Mukai
Y, Voss T, Furtek C, et al. Randomized trial of verubecestat for mild-tomoderate Alzheimer’s disease. N Engl J Med. 2018;378:1691–703.
7. Fleisher AS, Raman R, Siemers ER, Becerra L, Clark CM, Dean RA, Farlow
MR, Galvin JE, Peskind ER, Quinn JF, et al. Phase 2 safety trial targeting
amyloid beta production with a gamma-secretase inhibitor in Alzheimer disease. Arch Neurol. 2008;65:1031–8.
8. May PC, Dean RA, Lowe SL, Martenyi F, Sheehan SM, Boggs LN, Monk
SA, Mathes BM, Mergott DJ, Watson BM, et al. Robust central reductionof amyloid-beta in humans with an orally available, non-peptidic betasecretase inhibitor. J Neurosci. 2011;31:16507–16.
9. Siemers ER, Quinn JF, Kaye J, Farlow MR, Porsteinsson A, Tariot P,
Zoulnouni P, Galvin JE, Holtzman DM, Knopman DS, et al. Effects ofa gamma-secretase inhibitor in a randomized study of patients with
Alzheimer disease. Neurology. 2006;66:602–4.
10. Wessels AM, Lines C, Stern RA, Kost J, Voss T, Mozley LH, Furtek C,
Mukai Y, Aisen PS, Cummings JL, et al. Cognitive outcomes in trialsof two BACE inhibitors in Alzheimer’s disease. Alzheimers Dement.
2020;16:1483–92.
11. Podlesny-Drabiniok A, Marcora E, Goate AM. Microglial phagocytosis: adisease-associated process emerging from Alzheimer’s disease genetics.
Trends Neurosci. 2020;43:965–79.
12. Baik SH, Kang S, Son SM, Mook-Jung I. Microglia contributes to plaquegrowth by cell death due to uptake of amyloid beta in the brain of
Alzheimer’s disease mouse model. Glia. 2016;64:2274–90.
13. Doens D, Fernandez PL. Microglia receptors and their implications in the
response to amyloid beta for Alzheimer’s disease pathogenesis. J Neuroinflammation. 2014;11:48.
14. Paolicelli RC, Bolasco G, Pagani F, Maggi L, Scianni M, Panzanelli P,
Giustetto M, Ferreira TA, Guiducci E, Dumas L, et al. Synaptic pruning by microglia is necessary for normal brain development. Science.
2011;333:1456–8.
15. Gomez-Nicola D, Fransen NL, Suzzi S, Perry VH. Regulation of microglial proliferation during chronic neurodegeneration. J Neurosci.
2013;33:2481–93.
16. Koenigsknecht J, Landreth G. Microglial phagocytosis of fibrillar betaamyloid through a beta1 integrin-dependent mechanism. J Neurosci.
2004;24:9838–46.
17. Sasaki A, Yamaguchi H, Ogawa A, Sugihara S, Nakazato Y. Microglial
activation in early stages of amyloid beta protein deposition. Acta Neuropathol. 1997;94:316–22.
18. Liu B. Modulation of microglial pro-inflammatory and neurotoxic activity
for the treatment of Parkinson’s disease. AAPS J. 2006;8:E606-621.
19. Long-Smith CM, Sullivan AM, Nolan YM. The influence of microglia on thepathogenesis of Parkinson’s disease. Prog Neurobiol. 2009;89:277–87.
20. Hill KE, Zollinger LV, Watt HE, Carlson NG, Rose JW. Inducible nitric oxide
synthase in chronic active multiple sclerosis plaques: distribution, cellular expression and association with myelin damage. J Neuroimmunol.
2004;151:171–9.
21. Mack CL, Vanderlugt-Castaneda CL, Neville KL, Miller SD. Microglia are
activated to become competent antigen presenting and effector cells
in the inflammatory environment of the Theiler’s virus model of multiple
sclerosis. J Neuroimmunol. 2003;144:68–79.
22. Rose JW, Hill KE, Watt HE, Carlson NG. Inflammatory cell expression of
cyclooxygenase-2 in the multiple sclerosis lesion. J Neuroimmunol.
2004;149:40–9.
23. Krasemann S, Madore C, Cialic R, Baufeld C, Calcagno N, El Fatimy R, Beckers L, O’Loughlin E, Xu Y, Fanek Z, et al. The TREM2-APOE pathway drives
the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. Immunity. 2017;47:566-581 e569.
24. Norris GT, Smirnov I, Filiano AJ, Shadowen HM, Cody KR, Thompson JA,
Harris TH, Gaultier A, Overall CC, Kipnis J. Neuronal integrity and complement control synaptic material clearance by microglia after CNS injury. J
Exp Med. 2018;215:1789–801.

Page 17 of 19

25. Fu AK, Hung KW, Yuen MY, Zhou X, Mak DS, Chan IC, Cheung TH, Zhang
B, Fu WY, Liew FY, Ip NY. IL-33 ameliorates Alzheimer’s disease-like pathology and cognitive decline. Proc Natl Acad Sci U S A. 2016;113:E2705-2713.
26. Lee CYD, Daggett A, Gu X, Jiang LL, Langfelder P, Li X, Wang N, Zhao Y,
Park CS, Cooper Y, et al. Elevated TREM2 gene dosage reprograms microglia responsivity and ameliorates pathological phenotypes in Alzheimer’s
disease models. Neuron. 2018;97:1032-1048 e1035.
27. Nolte C, Moller T, Walter T, Kettenmann H. Complement 5a controls
motility of murine microglial cells in vitro via activation of an inhibitory
G-protein and the rearrangement of the actin cytoskeleton. Neuroscience. 1996;73:1091–107.
28. Uhlemann R, Gertz K, Boehmerle W, Schwarz T, Nolte C, Freyer D,
Kettenmann H, Endres M, Kronenberg G. Actin dynamics shape microglia
effector functions. Brain Struct Funct. 2016;221:2717–34.
29. Kunkle BW, Grenier-Boley B, Sims R, Bis JC, Damotte V, Naj AC, Boland A,
Vronskaya M, van der Lee SJ, Amlie-Wolf A, et al. Genetic meta-analysis
of diagnosed Alzheimer’s disease identifies new risk loci and implicates
Abeta, tau, immunity and lipid processing. Nat Genet. 2019;51:414–30.
30. Lambert JC, Ibrahim-Verbaas CA, Harold D, Naj AC, Sims R, Bellenguez C,
DeStafano AL, Bis JC, Beecham GW, Grenier-Boley B, et al. Meta-analysis
of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer’s
disease. Nat Genet. 2013;45:1452–8.
31. Beecham GW, Hamilton K, Naj AC, Martin ER, Huentelman M, Myers AJ,
Corneveaux JJ, Hardy J, Vonsattel JP, Younkin SG, et al. Genome-wide
association meta-analysis of neuropathologic features of Alzheimer’s
disease and related dementias. PLoS Genet. 2014;10: e1004606.
32. Jiao B, Liu X, Zhou L, Wang MH, Zhou Y, Xiao T, Zhang W, Sun R, Waye MM,
Tang B, Shen L. Polygenic analysis of late-onset Alzheimer’s disease from
Mainland China. PLoS ONE. 2015;10: e0144898.
33. Lawingco T, Chaudhury S, Brookes KJ, Guetta-Baranes T, Guerreiro R, Bras
J, Hardy J, Francis P, Thomas A, Belbin O, Morgan K. Genetic variants inglutamate-, Abeta-, and tau-related pathways determine polygenic risk
for Alzheimer’s disease. Neurobiol Aging. 2021;101:299 e213-299 e221.
34. Nettiksimmons J, Tranah G, Evans DS, Yokoyama JS, Yaffe K. Gene-based
aggregate SNP associations between candidate AD genes and cognitive
decline. Age (Dordr). 2016;38:41.
35. Wang X, Lopez O, Sweet RA, Becker JT, DeKosky ST, Barmada MM,
Feingold E, Demirci FY, Kamboh MI. Genetic determinants of survival inpatientswith Alzheimer’s disease. J Alzheimers Dis. 2015;45:651–8.
36. Menegon A, Burgaya F, Baudot P, Dunlap DD, Girault JA, Valtorta F. FAK+
and PYK2/CAKbeta, two related tyrosine kinases highly expressed in thecentral nervous system: similarities and differences in the expression pattern. Eur J Neurosci. 1999;11:3777–88.
37. Zhang C, Lambert MP, Bunch C, Barber K, Wade WS, Krafft GA, Klein WL.
Focal adhesion kinase expressed by nerve cell lines shows increased
tyrosine phosphorylation in response to Alzheimer’s A beta peptide. J
Biol Chem. 1994;269:25247–50.
38. Schaller MD. Calcium-dependent Pyk2 activation: a role for calmodulin?
Biochem J. 2008;410:e3-4.
39. Shen CJ, Raghavan S, Xu Z, Baranski JD, Yu X, Wozniak MA, Miller JS, Gupta
M, Buckbinder L, Chen CS. Decreased cell adhesion promotes angiogenesis in a Pyk2-dependent manner. Exp Cell Res. 2011;317:1860–71.
40. Rolon-Reyes K, Kucheryavykh YV, Cubano LA, Inyushin M, Skatchkov SN,
Eaton MJ, Harrison JK, Kucheryavykh LY. Microglia activate migration ofglioma cells through a Pyk2 intracellular pathway. PLoS ONE. 2015;10:e0131059.
41. Hide I, Shiraki H, Masuda A, Maeda T, Kumagai M, Kunishige N, Yanase Y,
Harada K, Tanaka S, Sakai N. P2Y(2) receptor mediates dying cell removal
via inflammatory activated microglia. J Pharmacol Sci. 2023;153:55–67.
42. Xi CX, Xiong F, Zhou Z, Mei L, Xiong WC. PYK2 interacts with MyD88 andregulates MyD88-mediated NF-kappaB activation in macrophages. J
Leukoc Biol. 2010;87:415–23.
43. Naser R, Aldehaiman A, Diaz-Galicia E, Arold ST. Endogenous control
mechanisms of FAK and PYK2 and their relevance to cancer development. Cancers. 2018;10:96.
44. Mastrolia V, Al Massadi O, de Pins B, Girault JA. Pyk2 in dorsal hippocampus plays a selective role in spatial memory and synaptic plasticity. Sci
Rep. 2021;11:16357.
45. Salazar SV, Cox TO, Lee S, Brody AH, Chyung AS, Haas LT, Strittmatter SM.
Alzheimer’s Disease risk factor Pyk2 mediates amyloid-beta-induced
synaptic dysfunction and loss. J Neurosci. 2019;39:758–72.

Lee et al. Journal of Neuroinflammation(2024) 21:196

46. Bartos JA, Ulrich JD, Li H, Beazely MA, Chen Y, Macdonald JF, Hell JW.
Postsynaptic clustering and activation of Pyk2 by PSD-95. J Neurosci.
2010;30:449–63.
47. Guo Y, Sun CK, Tang L, Tan MS. Microglia PTK2B/Pyk2 in the pathogenesisof Alzheimer’s disease. Curr Alzheimer Res. 2024;20:692–704.
48. Johnson RD, Schauerte JA, Wisser KC, Gafni A, Steel DG. Direct observation of single amyloid-beta(1–40) oligomers on live cells: binding andgrowth at physiological concentrations. PLoS ONE. 2011;6: e23970.
49. Ryan TM, Caine J, Mertens HD, Kirby N, Nigro J, Breheney K, Waddington
LJ, Streltsov VA, Curtain C, Masters CL, Roberts BR. Ammonium hydroxide
treatment of Abeta produces an aggregate free solution suitable forbiophysical and cell culture characterization. PeerJ. 2013;1: e73.
50. Condello C, Yuan P, Schain A, Grutzendler J. Microglia constitute a barrierthat prevents neurotoxic protofibrillar Abeta42 hotspots around plaques.
Nat Commun. 2015;6:6176.
51. Taylor ZV, Khand B, Porgador A, Monsonego A, Eremenko E. An optimized
intracerebroventricular injection of CD4(+) T cells into mice. STAR Protoc.
2021;2:100725.
52. Loryan I, Friden M, Hammarlund-Udenaes M. The brain slice method forstudying drug distribution in the CNS. Fluids Barriers CNS. 2013;10:6.
53. Pai KS, Shankar SK, Ravindranath V. A simple and inexpensive slicer for
preparation of brain slices. J Neurosci Methods. 1991;37:209–14.
54. Okigaki M, Davis C, Falasca M, Harroch S, Felsenfeld DP, Sheetz MP,
Schlessinger J. Pyk2 regulates multiple signaling events crucial for
macrophage morphology and migration. Proc Natl Acad Sci U S A.
2003;100:10740–5.
55. Imai Y, Kohsaka S. Intracellular signaling in M-CSF-induced microglia
activation: role of Iba1. Glia. 2002;40:164–74.
56. Marzolo MP, von Bernhardi R, Inestrosa NC. Mannose receptor is present
in a functional state in rat microglial cells. J Neurosci Res. 1999;58:387–95.
57. Cao LG, Wang YL. Mechanism of the formation of contractile ring in dividing cultured animal cells. II. Cortical movement of microinjected actin
filaments. J Cell Biol. 1990;111:1905–11.
58. Bornens M. The centrosome in cells and organisms. Science.
2012;335:422–6.
59. Wong C, Stearns T. Centrosome number is controlled by a centrosomeintrinsic block to reduplication. Nat Cell Biol. 2003;5:539–44.
60. Wang X, Rousset CI, Hagberg H, Mallard C. Lipopolysaccharide-induced
inflammation and perinatal brain injury. Semin Fetal Neonatal Med.
2006;11:343–53.
61. Lund S, Christensen KV, Hedtjarn M, Mortensen AL, Hagberg H, Falsig J,
Hasseldam H, Schrattenholz A, Porzgen P, Leist M. The dynamics of the
LPS triggered inflammatory response of murine microglia under different
culture and in vivo conditions. J Neuroimmunol. 2006;180:71–87.
62. Hoogland IC, Houbolt C, van Westerloo DJ, van Gool WA, van de Beek D.
Systemic inflammation and microglial activation: systematic review ofanimal experiments. J Neuroinflammation. 2015;12:114.
63. Streit WJ, Xue QS, Tischer J, Bechmann I. Microglial pathology. Acta Neuropathol Commun. 2014;2:142.
64. Salter MW, Stevens B. Microglia emerge as central players in brain disease.
Nat Med. 2017;23:1018–27.
65. Hampel H, Hardy J, Blennow K, Chen C, Perry G, Kim SH, Villemagne VL,
Aisen P, Vendruscolo M, Iwatsubo T, et al. The amyloid-beta pathway in
Alzheimer’s disease. Mol Psychiatry. 2021;26:5481–503.
66. DaRocha-Souto B, Scotton TC, Coma M, Serrano-Pozo A, Hashimoto T,
Sereno L, Rodriguez M, Sanchez B, Hyman BT, Gomez-Isla T. Brain oligomeric beta-amyloid but not total amyloid plaque burden correlates withneuronal loss and astrocyte inflammatory response in amyloid precursor
protein/tau transgenic mice. J Neuropathol Exp Neurol. 2011;70:360–76.
67. Jansen IE, Savage JE, Watanabe K, Bryois J, Williams DM, Steinberg S, Sealock J, Karlsson IK, Hagg S, Athanasiu L, et al. Genome-wide meta-analysis
identifies new loci and functional pathways influencing Alzheimer’s
disease risk. Nat Genet. 2019;51:404–13.
68. Kamboh MI, Demirci FY, Wang X, Minster RL, Carrasquillo MM, Pankratz VS,
Younkin SG, Saykin AJ, Alzheimer’s Disease Neuroimaging I, Jun G, et al.
Genome-wide association study of Alzheimer’s disease. Transl Psychiatry.
2012;2: e117.
69. Li YQ, Tan MS, Wang HF, Tan CC, Zhang W, Zheng ZJ, Kong LL, Wang
ZX, Tan L, Jiang T, et al. Common variant in PTK2B is associated withlate-onset Alzheimer’s disease: a replication study and meta-analyses.
Neurosci Lett. 2016;621:83–7.

Page 18 of 19

70. Haas LT, Strittmatter SM. Oligomers of amyloid beta prevent physiological activation of the cellular prion protein-metabotropic glutamate
receptor 5 complex by glutamate in Alzheimer disease. J Biol Chem.
2016;291:17112–21.
71. Kaufman AC, Salazar SV, Haas LT, Yang J, Kostylev MA, Jeng AT, Robinson
SA, Gunther EC, van Dyck CH, Nygaard HB, Strittmatter SM. Fyn inhibition
rescues established memory and synapse loss in Alzheimer mice. Ann
Neurol. 2015;77:953–71.
72. Brody AH, Strittmatter SM. Synaptotoxic signaling by amyloid betaoligomers in Alzheimer’s disease through prion protein and mGluR5. Adv
Pharmacol. 2018;82:293–323.
73. Brody AH, Nies SH, Guan F, Smith LM, Mukherjee B, Salazar SA, Lee S, Lam
TKT, Strittmatter SM. Alzheimer risk gene product Pyk2 suppresses tau
phosphorylation and phenotypic effects of tauopathy. Mol Neurodegener. 2022;17:32.
74. Lee S, Salazar SV, Cox TO, Strittmatter SM. Pyk2 Signaling through Graf1
and RhoA GTPase is required for amyloid-beta oligomer-triggered synapse loss. J Neurosci. 2019;39:1910–29.
75. Dickson DW. Multinucleated giant cells in acquired immunodeficiency
syndrome encephalopathy. Origin from endogenous microglia? Arch
Pathol Lab Med. 1986;110:967–8.
76. Michaels J, Price RW, Rosenblum MK. Microglia in the giant cell encephalitis of acquired immune deficiency syndrome: proliferation, infection andfusion. Acta Neuropathol. 1988;76:373–9.
77. Kreutzberg GW. Microglia: a sensor for pathological events in the CNS.
Trends Neurosci. 1996;19:312–8.
78. Hornik TC, Neniskyte U, Brown GC. Inflammation induces multinucleationof microglia via PKC inhibition of cytokinesis, generating highly phagocytic multinucleated giant cells. J Neurochem. 2014;128:650–61.
79. Peterson PK, Gekker G, Hu S, Anderson WB, Teichert M, Chao CC, Molitor
TW. Multinucleated giant cell formation of swine microglia induced by
Mycobacterium bovis. J Infect Dis. 1996;173:1194–201.
80. Ahmadzadeh K, Vanoppen M, Rose CD, Matthys P, Wouters CH. Multinucleated giant cells: current insights in phenotype, biological activities,
and mechanism of formation. Front Cell Dev Biol. 2022;10:873226.
81. Valimaki E, Miettinen JJ, Lietzen N, Matikainen S, Nyman TA. Monosodium
urate activates Src/Pyk2/PI3 kinase and cathepsin dependent unconventional protein secretion from human primary macrophages. Mol Cell
Proteomics. 2013;12:749–63.
82. Combs CK, Johnson DE, Cannady SB, Lehman TM, Landreth GE. Identification of microglial signal transduction pathways mediating a neurotoxic
response to amyloidogenic fragments of beta-amyloid and prion proteins. J Neurosci. 1999;19:928–39.
83. Belrose JC, Jackson MF. TRPM2: a candidate therapeutic target for treatingneurological diseases. Acta Pharmacol Sin. 2018;39:722–32.
84. Yamamoto S, Shimizu S, Kiyonaka S, Takahashi N, Wajima T, Hara Y, Negoro
T, Hiroi T, Kiuchi Y, Okada T, et al. TRPM2-mediated Ca2+influx induces
chemokine production in monocytes that aggravates inflammatory
neutrophil infiltration. Nat Med. 2008;14:738–47.
85. Alawieyah Syed Mortadza S, Sim JA, Neubrand VE, Jiang LH. A criticalrole of TRPM2 channel in Abeta(42) -induced microglial activation andgeneration of tumor necrosis factor-alpha. Glia. 2018;66:562–75.
86. Ryzhakov G, Almuttaqi H, Corbin AL, Berthold DL, Khoyratty T, Eames HL,
Bullers S, Pearson C, Ai Z, Zec K, et al. Defactinib inhibits PYK2 phosphorylation of IRF5 and reduces intestinal inflammation. Nat Commun.
2021;12:6702.
87. Corsi JM, Rouer E, Girault JA, Enslen H. Organization and post-transcriptional processing of focal adhesion kinase gene. BMC Genomics.
2006;7:198.
88. Hall JE, Fu W, Schaller MD. Focal adhesion kinase: exploring Fak structure
to gain insight into function. Int Rev Cell Mol Biol. 2011;288:185–225.
89. Schaller MD. Cellular functions of FAK kinases: insight into molecularmechanisms and novel functions. J Cell Sci. 2010;123:1007–13.
90. Wu Y, Li N, Ye C, Jiang X, Luo H, Zhang B, Zhang Y, Zhang Q. Focal adhesion kinase inhibitors, a heavy punch to cancer. Discov Oncol. 2021;12:52.
91. Jeon M, Hong S, Cho H, Park H, Lee SM, Ahn S. Targeting FAK/PYK2 withSJP1602 for anti-tumor activity in triple-negative breast cancer. Curr
Issues Mol Biol. 2023;45:7058–74.
92. Saito T, Matsuba Y, Mihira N, Takano J, Nilsson P, Itohara S, Iwata N, Saido
TC. Single app knock-in mouse models of Alzheimer’s disease. Nat Neurosci. 2014;17:661–3.

Lee et al. Journal of Neuroinflammation(2024) 21:196

93. Sato K, Watamura N, Fujioka R, Mihira N, Sekiguchi M, Nagata K, Ohshima
T, Saito T, Saido TC, Sasaguri H. A third-generation mouse model of
Alzheimer’s disease shows early and increased cored plaque pathology composed of wild-type human amyloid beta peptide. J Biol Chem.
2021;297:101004.
94. Giralt A, de Pins B, Cifuentes-Diaz C, Lopez-Molina L, Farah AT, Tible M,
Deramecourt V, Arold ST, Gines S, Hugon J, Girault JA. PTK2B/Pyk2 overexpression improves a mouse model of Alzheimer’s disease. Exp Neurol.
2018;307:62–73.
95. Kilinc D, Vreulx AC, Mendes T, Flaig A, Marques-Coelho D, Verschoore M,
Demiautte F, Amouyel P, Neuro CEBBB, Eysert F, et al. Pyk2 overexpressionin postsynaptic neurons blocks amyloid beta(1–42)-induced synaptotoxicity in microfluidic co-cultures. Brain Commun. 2020;2:fcaa139.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Page 19 of 19