(2024) 21:125
Li et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03119-2

Journal of Neuroinflammation

Open Access

RESEARCH

Monocytes release cystatin F dimer
to associate with Aβ and aggravate amyloid
pathology and cognitive deficits in Alzheimer’s
diseaseQiang Li1,2†, Bing Li1†, Li Liu3,4†, Kang‑Ji Wang1†, Ming‑Yue Liu1, Yu Deng5, Ze Li1, Wei‑Dong Zhao1*,
Li‑Yong Wu3*, Yu‑Hua Chen1* and Ke Zhang1*

Abstract
Background Understanding the molecular mechanisms of Alzheimer’s disease (AD) has important clinical impli‑
cations for guiding therapy. Impaired amyloid beta (Aβ) clearance is critical in the pathogenesis of sporadic AD,
and blood monocytes play an important role in Aβ clearance in the periphery. However, the mechanism underlying
the defective phagocytosis of Aβ by monocytes in AD remains unclear.
Methods Initially, we collected whole blood samples from sporadic AD patients and isolated the monocytes for RNA
sequencing analysis. By establishing APP/PS1 transgenic model mice with monocyte-specific cystatin F overexpres‑sion, we assessed the influence of monocyte-derived cystatin F on AD development. We further used a nondenatur‑ing gel to identify the structure of the secreted cystatin F in plasma. Flow cytometry, enzyme-linked immunosorbent
assays and laser scanning confocal microscopy were used to analyse the internalization of Aβ by monocytes. Pull
down assays, bimolecular fluorescence complementation assays and total internal reflection fluorescence micros‑
copy were used to determine the interactions and potential interactional amino acids between the cystatin F
protein and Aβ. Finally, the cystatin F protein was purified and injected via the tail vein into 5XFAD mice to assess AD
pathology.
Results Our results demonstrated that the expression of the cystatin F protein was specifically increasedin the monocytes of AD patients. Monocyte-derived cystatin F increased Aβ deposition and exacerbated cogni‑
tive deficits in APP/PS1 mice. Furthermore, secreted cystatin F in the plasma of AD patients has a dimeric structurethat is closely related to clinical signs of AD. Moreover, we noted that the cystatin F dimer blocks the phagocytosis†

Qiang Li, Bing Li, Li Liu and Kang-Ji Wang have contributed equally to thiswork.
*Correspondence:
Wei‑Dong Zhao
wdzhao@cmu.edu.cn
Li‑Yong Wu
wmywly@hotmail.com
Yu‑Hua Chenyhchen@cmu.edu.cn
Ke Zhangkzhang@cmu.edu.cn
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise 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 thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom‑mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Li et al. Journal of Neuroinflammation(2024) 21:125

Page 2 of 20

of Aβ by monocytes. Mechanistically, the cystatin F dimer physically interacts with Aβ to inhibit its recognition
and internalization by monocytes through certain amino acid interactions between the cystatin F dimer and Aβ.
We found that high levels of the cystatin F dimer protein in blood contributed to amyloid pathology and cognitive
deficits as a risk factor in 5XFAD mice.
Conclusions Our findings highlight that the cystatin F dimer plays a crucial role in regulating Aβ metabolism via its
peripheral clearance pathway, providing us with a potential biomarker for diagnosis and potential target for therapeu‑
tic intervention.
Keywords Alzheimer’s disease, Amyloid beta, Cystatin F, Monocyte, Peripheral clearance

Background
Alzheimer’s disease (AD) is the most common form ofdementia and an age-related neurodegenerative diseasecharacterized by extracellular amyloid plaques, intracellular neurofibrillary tangles, and neuroinflammation
triggered by activated microglia and reactive astrocytes[1, 2]. Accumulating evidence supports the idea that an
imbalance between the production and elimination ofamyloid beta (Aβ) is a very early sign of AD pathogenesis[3, 4]. Familial AD (early-onset AD), which is caused by
the overproduction of Aβ due to mutations in the amyloid precursor protein (APP) and PSEN1 (PS1) genes,
accounts for only 1% of all AD cases [5, 6]. However, insporadic AD (late-onset AD), which represents 99% of all
AD patients, impaired Aβ clearance is thought to be theleading cause of Aβ accumulation. Metabolic labellingstudies have shown that Aβ clearance is impaired while
Aβ production remains unchanged in sporadic late-onset
AD [7]. Therefore, exploring dysfunctional molecular andcellular mechanisms involved in the clearance of Aβ will
enhance the understanding of AD pathophysiology andpromote the development of new clinical interventions.
Aβ is the product of the proteolytic cleavage of APP
and is mainly expressed in neurons [8, 9]. Traditionally,Aβ has been thought to be cleared primarily by cells inthe brain [10, 11]. For instance, microglia, the major
type of innate immune cell in the brain, can eliminate
various forms of Aβ through cell-mediated phagocytosis and extracellular enzymatic degradation [12, 13].
However, convincing evidence has indicated that the
peripheral system plays a role in the clearance of Aβ[14–17]. Approximately 40–60% of brain-derived Aβ
is estimated to be transported to the peripheral systemfor clearance [17, 18]. Blood monocytes are the counterparts of microglia in the periphery [19, 20]. Monocytes/macrophages are vital members of the peripheral
innate immune system and act as the first line of hostdefence through multiple effector functions [21]. Blood
monocytes clear Aβ that is transported from the brain
and slow the progression of AD [22]. Some studies haveshown that monocytes are more effective than resident
microglia at providing neuroprotection, regulatingneuroinflammation, and clearing Aβ in AD [18, 19, 23–25]. However, monocytes derived from patients withAD display an unexplainable degree of phagocytic dysfunction; for example, macrophages from the majority of AD patients fail to transport Aβ into endosomes
and lysosomes [25, 26], monocytes inefficiently clear
Aβ from regions of the AD brain [27], and the expression of phagocytosis-related receptors was decreased inmonocytes from AD patients [28]. Therefore, the determinants of Aβ elimination by monocytes may be potential interventional targets for the peripheral clearanceof Aβ.
Cystatin F, encoded by the Cst 7 gene, is a secreted
protein that is specifically expressed in immune cells
such as monocytes/macrophages, lymphocytes, andneutrophils in the peripheral circulation and exclusively in microglia in the central nervous system. Cystatin F is most likely involved in the immune response[29–34]. When cystatin F is synthesized or taken up
intracellularly, it enters the endosome-lysosome compartment, where it acts as an endogenous inhibitorof cysteine proteases such as cathepsin L and C [35].
Recently, cystatin F has been regarded as a diseaseassociated microglia (DAM) signature in AD that regulates microglial phagocytosis via an unclear mechanism[36, 37]. However, the role of cystatin F, especially its
secreted form in peripheral monocytes, remains largely
unknown.
Here, we identified increased mRNA expression ofcystatin F in monocytes isolated from patients with AD.
Monocyte-derived cystatin F exacerbate Aβ depositionin the brain and cognitive impairment in APP/PS1 mice.
Mechanistically, we found that cystatin F was releasedby monocytes as a dimer into the plasma and physically interacted with Aβ to inhibit its internalization by
monocytes. High-level cystatin F dimers in plasma rapidly aggravate cognitive impairment in 5XFAD transgenic mice, suggesting that circulatory cystatin F inhibits
peripheral Aβ clearance and leads to deteriorated Aβ
deposition in the brain, providing us with a potential therapeutic target for the elimination of Aβ in the
periphery.

Li et al. Journal of Neuroinflammation(2024) 21:125

Methods
Patients and clinical assessment

Age-matched controls and AD patients were recruitedfrom Xuanwu Hospital of Capital Medical University andFirst Hospital of China Medical University (sTable 1).
The National Institute of Neurological and Communication Disorders and the Stroke and Alzheimer’s Disease
and Related Disorders Association (NINCDS-ADRDA)
were used as the basis for the patient selection criteria,
and patients with other types of dementia were excluded
from this study [38]. All of the healthy control individuals had no substantial comorbidities that could impair
brain function and no family history of dementia, and thepatients with AD did not have immunological illnesses or
vascular risk factors [39]. The Mini-Mental State Examination (MMSE), the Montreal Cognitive Assessment(MoCA) [40], and the Clinical Dementia Rating (CDR)[41, 42] were all used to measure cognitive function
and the degree of impairment in all subjects. To evaluate object recognition memory, the Rey Auditory Verbal
Learning Test (RAVLT) was used as described previously[43]. All participants (or their legal guardians) provided
written informed consent.
Isolation of plasma, monocytes, lymphocytes
and neutrophils

Approximately 5 mL of peripheral whole blood was
extracted from each individual by venipuncture. The
plasma was extracted from the whole blood by centrifuging for 10 min at 800 ×g. The monocytes and lymphocytes
were separated by using Dynabeads Isolation Reagent(Invitrogen, Carlsbad, CA, Cat No. 11145D;11149D)
according to the manufacturer’s instructions. Neutrophils were isolated by using Percoll (Sigma Aldrich, MO,
USA, Cat No. P1644) density gradient centrifugation.
RNA sequencing and data analysis

The RNA of the primary monocytes from the AD patientsand age-matched controls was extracted by using TRIzol
reagent (Invitrogen, Carlsbad, CA, Cat No. 15596026CN)
according to the instructions. RNA sequencing analysis was carried out by Biomarker Technologies Company (Beijing, China). For data analysis, the fold change(FC), an absolute fold change between the two groups foreach transcript, was calculated when comparing the twogroups with distinct profiles. A Student’s t test was used
to determine the statistical significance of the differences
between groups. The significantly differentially expressedtranscripts had a FC of at least 4 and p values less than

Page 3 of 20

0.05. By dividing the total number of genes in the Gene
Ontology (GO) category by the total number of differentially expressed genes, enrichment factors were computed. Gene Set Enrichment Analysis (GSEA) v2.0.14
software was used to perform GSEA analysis.
Mice

APP/PS1 transgenic mice were obtained from China
Medical University. 5 × FAD transgenic mice were generous gifts from Professor Chao Wang of Chongqing
Medical University. Monocyte-specific human cystatin F-overexpressing transgenic mice were generated as
described in previous reports [44, 45]. Briefly, the open
reading frame (ORF) of human cystatin F was obtained
and subcloned and inserted into pBluescript SK (-) under
the control of the human CD68 promoter. The fragment
was removed and injected into fertilized C57BL/6 mice
to construct transgenic mice named Hmo-cys ­F+ mice.
Hmo-cys ­F+ and APP/PS1 AD model mice were bred to
produce APP/PS1/Hmo-cys ­F+ double transgenic mice.
All transgenic lines were backcrossed to the C57BL/6J
strain. Mice were housed under specific pathogen-free
conditions on a 12 h light/12 h dark cycle with freeaccess to water and food and no more than five mice per
cage. The temperature and humidity were 18–29 °C and
45–55%, respectively. In this study, only male 9-monthold APP/PS1 mice and 3-month-old 5XFAD mice were
included. All the animal experimental procedures wereapproved by the Animal Experimentation Ethics Committee of China Medical University (CMU20240091).
Morris water maze (MWM) test

The spatial memory and learning abilities of the mice
were assessed by the MWM test. One day before theexperiment, the mice were allowed to become accustomed to the water maze (100 cm in diameter). The pool
was filled with water that was rendered opaque, which
was drained every day, and the temperature of the water
was maintained at 19 °C to 22 °C. During the training
period, the mice were permitted to swim freely for 60 s tolocate the platform (9 cm in diameter), which was fixed at
a depth of 1 cm below the surface of the water. Mice that
did not find the platform were directed to it and allowed
to rest for 30 s. The mice were trained four times per day
for six days. On day seven, the platform was removed,
and the swimming activity of each mouse, including thelatency, number of platform crossings and velocity within
1 min, was recorded. All the processes were monitored
by using a video camera mounted overhead and automatically recorded via ANY-maze behavioural tracking software (Stoelting, Wood Dale, IL, USA).

Li et al. Journal of Neuroinflammation(2024) 21:125

Novel object recognition (NOR) test

The NOR test was conducted as described previously[46]. Initially, the mice were acclimated to the testing room for five days before testing. Then, the mice
were allowed to explore freely in the empty arena(40 × 40 × 40 cm) for 10 min. For the training session,
two identical cylinders (6 cm in diameter) were placedin the arena 5 cm away from the wall. The mice wereindividually placed in the arena and allowed to freely
explore for 10 min. Twenty-four hours after the training session, the NOR test was carried out. One of thefamiliar cylinders was replaced with a cube (with a side
length of 6 cm). The mice were allowed to explore for10 min, and a camera tracking system (TopScan Suite,
CleverSys Inc., Reston, VA, USA) was used to record theamount of time the mice spent exploring the objects.
The recognition index = time spent exploring the new
object/(time spent exploring the new object + timespent exploring the familiar object) × 100%.
Tail intravenous injections

Three-month-old 5xFAD mice were given tail intravenous injections of murine cystatin F dimer protein at adose of 200 μg/kg every three days for one month. The
experiments were performed 48 h after the last injection.
Tissue preparation and immunofluorescence

Briefly, mice were transcardially perfused with phosphate-buffered saline (PBS) followed by ice-cold 4% paraformaldehyde (PFA) in PBS (pH 7.4). After perfusion,
the brain was removed and postfixed in 4% PFA in PBS
at 4 °C overnight and then transferred to 30% sucrosefor 2-3 days until it sank to the bottom of the container.
Coronal sections of the brain (30 μm) were cut using acryostat (Minux FS800A, RWD Inc., China), collected
serially on coated glass slides, and stored at − 20 °C until
use. Six sections obtained from each brain containing
the cortex and CA1 area of the hippocampus were chosen for immunofluorescence staining. The brain sections
were incubated with PBS containing 0.5% Triton X-100
for 10 min and 5% BSA for 1 h at room temperature. The
brain sections were sequentially incubated overnightat 4 °C with primary antibodies recognizing Aβ (1:800;
Cell Signaling Technology, MA, USA, Cat No. 8243S),
His tag (1:500; ABclonal Technology, Wuhan, China,
Cat No. AE003) and IBAI (1:1000; Abcam, Cambs, UK,
Cat No. ab178846). Then, the sections were incubatedwith Alexa Fluor 488-conjugated donkey-anti-rabbit IgG(1:200; Invitrogen, CA, USA, Cat No. A-21206) for 3 h in
the dark at room temperature. After counterstaining with
4′,6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich,

Page 4 of 20

MO, USA, Cat No. 28718-90-3) for 5 min, the coverslips
were mounted and the slides were analysed under a laser
scanning confocal microscope (LSCM).
Enzyme‑linked immunosorbent assay (ELISA)

The levels of soluble and insoluble Aβ in the prefrontal
cortex were measured by ELISA method. Briefly, fresh
brain tissues from the mice were homogenized in RIPA
buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40,
and 0.5% sodium deoxycholate] supplemented with protease inhibitor cocktail, followed by centrifugation at
12,000 × g for 30 min at 4 °C. The supernatant was collected as the RIPA fraction, and the pellet was solubilized in 2% SDS and 25 mM Tris-HCl (pH 7.4) followedby centrifugation at 12,000 × g for 30 min at 4 °C as the
SDS fraction. The concentrations of Aβ40 and Aβ42 were
measured using ELISA kits (Elabscience Biotechnology
Company, Wuhan, China, Cat No. E-EL-M3009; E-ELM3010) according to the manufacturer’s instructions.
The Aβ40 and Aβ42 in human plasma were also determined by the ELISA kits (Elabscience Biotechnology
Company, Wuhan, China, Cat No. E-EL-H0542; E-ELH0543). In addition, the levels of cystatin F in the plasmaof patients with AD or AD mice were quantitatively
analysed by ELISA kits (CUSABIO Company, Wuhan,
China, Cat No. CSB-E17504h; CSB-EL006095MO). The
data were normalized to the total protein concentration.
Real‑time polymerase chain reaction (RT‑qPCR)

M-MLV reverse transcriptase was used to reverse
transcribe the total RNA from the cells. RT-qPCR
was conducted using a SYBR Premix Ex Taq Kit(Vazyme,Nanjing,China, Cat No. Q321-02) and an ABI
7500 RT-PCR instrument. Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was selected as the reference
gene. The conditions for PCR were 95 °C for 10 s, followed by 40 cycles of 95 °C for 30 s and 60 °C for 30 s.
Standard curves were established to calculate target genetranscript quantities in each sample.
Western blot analysis

The plasma protein concentrations were determined byusing a nondenaturing gel. Briefly, the plasma was diluted
with 1X PBS, and nondenaturing loading buffer (50%glycerol, 0.3 mol/L pH = 6.8 Tris-HCl, 10% SDS, 0.01%
bromophenol blue) was added to the samples. To detect
the monomeric form of cystatin F, loading buffer with
20% dithiothreitol (DTT) (Sangon biotech, shanghai,
China, Cat No. A620058) was used, and the samples wereboiled at 95 °C for 10 min. Coomassie Brilliant Blue (CBB)
staining of the plasma proteins served as an internal control. Cells were lysed with radioimmunoprecipitation

Li et al. Journal of Neuroinflammation(2024) 21:125

assay lysis buffer (Beyotime Biotech, Beijing, China, Cat
No. P0013B) containing 1 mM PMSF (Beyotime Biotech,
Beijing, China, Cat No.ST506). Then, the protein samples were separated on 12% SDS polyacrylamide gels.
For western blot detection, the primary antibody against
cystatin F (ABclonal Technology, Wuhan, China, Cat
No. A8164) was diluted 1:1000, and Horseradish Peroxidase (HRP) goat anti-rabbit IgG (ABclonal Technology,
Wuhan, China, Cat No. AS014) was diluted 1:10,000.
Immunoreactive bands were visualized with the SuperSignal West Pico chemiluminescent substrate (Pierce, IL,
USA) using an LAS-3000 mini (Fujifilm, Tokyo, Japan).
For quantitative analysis, the mean density of each band
was measured by ImageJ software.
Recombinant protein expression and purification

The ORF of cystatin F was subcloned and inserted into
the pcDNA3.1 (+) myc-His A vector, and the recombinant plasmids were transfected into 293T cells with
polyethylenimine (PEI) (Polysciences, IL, USA, Cat No.
02371-500) at a ratio of 1 µg of DNA to 2.5 µg of PEI[47, 48]. The cell culture supernatant was collected after
48 and 72 h of transfection, and the secreted cystatin F
protein was purified by using His-tag Purification Resin(Beyotime, Shanghai, China, Cat No. P2210).
Aβ internalization assay

The Aβ internalization by monocytes were assessedby flow cytometry analysis (FCM), ELISA and LSCM
methods. For FCM and ELSIA assay, 3 × ­105 cells perwell were cultured in 24-well plates, and the cystatin F
dimer protein was added at final concentrations of 0 ng/
mL, 50 ng/mL, 250 ng/mL, and 1000 ng/mL for 30 min.
Then a final concentration of 1 μg/mL Aβ42-Alexa Fluor
647 (Kaneka Eurogentec, Liege Province, Belgium, Cat
No. AS-64161) or soluble Aβ42 was added for a further
incubation for 30 min at 37 °C, after which the cells were
subjected to FCM and ELISA analysis, respectively. For
LSCM analysis, cells were pretreated with the cystatin F
dimer at a final concentration of 250 ng/mL for 30 min
and then cells were incubated with Aβ42-Alexa Fluor 555(Kaneka Eurogentec, Liege Province, Belgium, Cat No.
AS-60480-01) for an additional 30 min at 37 °C. To analysis the binding ability of cystatin F dimer to Aβ at thecell membrane by FCM and ELISA methods, cells werepretreated with the cystatin F dimer at a final concentration of 250 ng/mL for 30 min at 37 °C, and then cells wereincubated with Aβ42-Alexa Fluor 647 or soluble Aβ42 foran additional 30 min at 0 °C, after which the cells were
subjected to FCM and ELISA analysis, respectively. For
LSCM analysis, cells were pretreated with the cystatin F
dimer at a final concentration of 250 ng/mL for 30 min
at 37 °C, and then cells were incubated with Aβ42-Alexa

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Fluor 555 for an additional 30 min at 0 °C. The coverslips
were labelled with 15 μg/mL 3,3′-dioctadecyloxacarbocyanine perchlorate (DiOC18(3), Beyotime, Shanghai,
China, Cat No. C1038) in 4% PFA solution for 15 min,and stained with DAPI at a final concentration of 1 μg/
mL for 4 min.
For the uptake inhibition assay, cytochalasin D (GLPBIO, CA, USA, Cat No. D22914GC13440) or EIPA (MedChemExpress, NJ, USA, Cat No. HY-101840) at final
concentrations of 5 μg/mL or 40 μM were added to thecells to pretreat for 30 min and 1 h, respectively. The cells
were incubated with the cystatin F dimer at a final concentration of 250 ng/mL for 30 min. Aβ42-Alexa Fluor
647 at a final concentration of 1 μg/mL was added to thecells, which were incubated for 30 min and analysed via
FCM method. The data were analysed with InCyte software (Millipore, Darmstadt, Germany) and visualizedusing FlowJo software (Tree Star, Inc., CA, USA).
Total internal reflection fluorescence microscopy (TIRFM)

Monocytes were cultured on coverslips, and after treatment with 250 ng/mL cystatin F protein for 30 min, thecells were incubated with 1 μg/mL soluble Aβ42-Alexa
Fluor 555 for 30 min at 0 °C and washed three times with
PBS. The monocytes on the coverslips were fixed with
4% PFA solution for 15 min and were analysed under a
TIRFM.
Binding assay

The 96-well ELISA plates were coated with 4 ng/μL of
dissolved Aβ1-40, Aβ1-42, Aβ40-1, or Aβ42-1 for 16 h
at 4 °C. Bull serum albumin at a 5% concentration wasused as a blocking agent and incubated for 2 h at 37 °C.
Recombinant cystatin F dimer protein was added tothe plates for 3 h at various concentrations (18.75 ng/
mL, 37.5 ng/mL, 75 ng/mL, 150 ng/mL, and 300 ng/mL). Cystatin F antibody (R&D Systems, MN, USA,Cat No. MAB1889) was used to identify the binding of
the two proteins. A secondary antibody coupled to HRP
and 3,3′,5,5′-Tetramethylbenzidine reagent was used toinduce a colour reaction, and the absorbance was read at
450 nm.
Molecular docking

Protein Aβ42 (PDB ID: 6SZF) and cystatin F dimer (PDB
ID: 2CH9) crystal structures were retrieved from the
RCSB Protein Data Bank (http://​www.​rcsb.​org/). The
protein cystatin F was set as the receptor before docking,while Aβ42 was set as the ligand. Cluspro1, HDOCK2,
and MOE3 were utilized to determine the binding
mechanism of Aβ42 and the cystatin F dimer using threemolecular docking tools.

Li et al. Journal of Neuroinflammation(2024) 21:125

Pull‑down assay

Purified GST-Aβ protein (Abcam, Cambridge, UK) andHis-tag cystatin F were co-incubated overnight at 4 °C.
The purification resin (Beyotime, Shanghai, China, Cat
No. P2251; P2210) for the GST-tag or His-tag was added
to the protein mixture and incubated for 2 h at 4 °C. The
resins were centrifuged for 5 min at 1000 ×g and the precipitate was mixed with loading buffer, then the samples
were subjected to Western blot analysis. Rabbit antiHis-tag and mouse anti-GST-tag (ABclonal Technology,
Wuhan, China, Cat No. AE086; AE001) were diluted
1:1000. Goat anti-rabbit IgG or goat anti-mouse IgG conjugated with HRP was diluted 1:10,000 (ABclonal Technology, Wuhan, China, Cat No. AS070; AS066).
Bimolecular fluorescence complementation assay (BiFC)

The BiFC was carried out as previously reported withmodifications [49]. Briefly, full-length ORF of cystatin F
conjugated with the N-terminal fragment Venus and ofAβ conjugated with the C-terminal fragment Venus wereconstructed. The recombinant plasmids were transfected
into 293T cells, and the protein supernatants were purified. The two proteins were incubated at 200 ng/mL for
2 h at 37 °C, and the fluorescence intensity was detected
at 529 nm.
Statistical analysis

Data were presented as mean ± standard deviation (SD).
Student’s t test was used to compare two groups while
one-way ANOVA was used to compare more than two
groups. A receiver operating characteristic (ROC) curve
was generated to evaluate the diagnostic value of the cystatin F in AD monocytes. Correlations were assessed byusing Pearson correlation coefficient. All data were analysed by GraphPad Prism 8.0 statistical software. Statistical significance was indicated by a probability (p) value ofless than or equal to 0.05.

Results
Elevated cystatin F expression in monocytes isolated
from patients with AD

First, we collected samples from sporadic AD patientsand isolated monocytes for RNA-seq analysis (sTable 1
and sFig. 1A). We performed a GO functional enrichment analysis of the differentially expressed genes withfour fold changes in expression (Fig. 1A and sFig. 1B
and C). Since monocytes are the main peripheral innate
immune cells involved in the immune response andclearance of pathogenic agents, we selected the GO
terms “immune response” (GO:0006955), “immune
system process” (GO:0002376), and “cell activation”(GO:0001775) from the top 10 GO pathways to perform a GSEA analysis of these differentially expressed

Page 6 of 20

genes (Fig. 1B). There were 19 core enriched terms in
the three pathways (Fig. 1C and D). Among them, only
four enrichments were specific to immune cells (sTable 2). Ultimately, we chose the cystatin F protein asa target because it is expressed in myeloid leukaemia
cells involved in the innate immune response (Fig. 1D
and sTable 2). Cystatin F also has been reported to be
expressed in microglia, which are central innate immune
cells [50, 51]. Next, we examined the mRNA level of cystatin F in monocytes isolated from AD patients by RTqPCR with an expanded sample size (n = 40; sTable 1).
The results showed that the mRNA expression of cystatin F was obviously greater in the AD group than in theelderly control group (Fig. 1E). Neutrophils and lymphocytes are the main types of immune cells that participate
in innate and adaptive immune responses, respectively.
We also examined the mRNA expression of cystatin F
in the two types of immune cells. The results indicatedthat the transcript level was not significantly different between the AD group and age-matched controls(Fig. 1E). Furthermore, the specificity and sensitivity ofthe mRNA expression of cystatin F in AD monocytes
were analysed, and ROC analysis indicated that the area
under the curve (AUC) reached 0.8513, which was statistically significant (Fig. 1F). These results demonstrated
that aberrant expression of cystatin F principally occurs
in AD monocytes.
Overexpression of human cystatin F in monocytes
aggravated Aβ deposition and cognitive decline in APP/
PS1 mice

To explore the role of cystatin F in monocytes in the context of AD, we generated transgenic mice on the C57BL/6 background with monocyte-specific expression of human cystatin F under thehuman CD68 promoter (Hmo-cys ­F+) (Fig. 2A) [44, 45].
Hmo-cys ­F+ mice were hybridized with APP/PS1 mice toobtain APP/PS1/Hmo-cys ­F+ mice via double transgenics to mimic the high cystatin F levels observed in the
monocytes of AD patients (Fig. 2B-D). First, we detected
Aβ deposition and measured the Aβ concentration in thebrain. The Aβ plaque coverage within the brains of APP/
PS1/Hmo-cys ­F+ mice were markedly greater than thatwithin the brains of APP/PS1 mice according to immunofluorescence staining (Fig. 2E). Additionally, there wasa significant increase in soluble Aβ40 and Aβ42 levels(Fig. 2F, G) and insoluble Aβ40 and Aβ42 levels (Fig. 2H,I) in the brains of APP/PS1/Hmo-cys F­ + mice. Furthermore, we assessed the spatial learning and memory of
the mice through the MWM test. Compared with APP/
PS1 mice, APP/PS1/Hmo-cys F­ + mice showed significant spatial learning and memory deficits, as reflected bylonger escape latency time during the training trials and a

Li et al. Journal of Neuroinflammation(2024) 21:125

Page 7 of 20

Fig. 1 The mRNA expression of cystatin F was significantly increased in the monocytes of patients with AD. Monocytes derived from AD patients(n = 9) and age-matched controls (n = 9) were collected for RNA-sequencing analysis. A GO enrichment analysis of biological processes showing
the top 10 signalling pathways. B GSEA enrichment analysis of genes co-expressed in the three GO pathways. C Venn diagram of the genes
co-expressed in the three GO pathways. D Heatmap showing the 19 differentially expressed genes. E The mRNA expression of cystatin F
in monocytes from AD patients (n = 40) and controls (n = 40), and in lymphocytes (n = 20) and neutrophils (n = 20) from AD patients and controls(n = 20) was measured by RT-qPCR. F ROC curve analysis of cystatin F mRNA expression in monocytes from AD patients. The data are presented
as the means ± SD and were analysed using Student’s t test. ****p < 0.0001

Li et al. Journal of Neuroinflammation(2024) 21:125

lower number of platform crossings (Fig. 2J-L, N). However, there was no significant alterations in swimming
speed (Fig. 2M). In addition, the levels of soluble Aβ40
and Aβ42 were significantly increased in the plasma of
APP/PS1/Hmo-cys ­F+ transgenic mice compared with
APP/PS1 mice (Fig. 2O, P). These results suggested thathuman cystatin F specifically aggravated Aβ depositionin the brain and cognitive impairment in AD model mice.
Increased cystatin F dimer levels in the plasma of AD
patients

Mature cystatin F, which contains a signal peptide, is synthesized, and secreted into the extracellular space of cultured cells [50, 51]. Plasma is a major extracellular fluid
makes up 55% of the peripheral blood, and the presenceof cystatin F in plasma has not yet been investigated.
Therefore, we used a nondenaturing gel to analyse theexpression of cystatin F in the plasma of patients with
AD. As shown in Fig. 3A, the level of cystatin F, which isapproximately 50 kDa in molecular weight (MW), in theplasma from patients with AD was significantly greaterthan that in the plasma from controls. Cystatin F dimerizes through two disulfide bridges involving Cys26 on one
subunit and Cys63 on the other [35]. We selected DTT, areducing agent that can disrupt disulfide bonds to reduce
dimers to monomers, to determine the structure of cystatin F in plasma. We found that the MW of cystatin Fchanged to approximately 25 kDa when DTT was added,
suggesting that cystatin F in human plasma is a dimer inthe physiological state (Fig. 3B). Furthermore, we examined the level of native cystatin F in cultured monocytes.
Using a nondenaturing gel, the results showed that the
intracellular cystatin F protein remains predominantly
dimeric with few monomers and the extracellular proteins remained absolutely dimeric (sFig. 2). This finding
suggested that the level of secreted cystatin F significantly increased in AD plasma as dimeric structure.

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Next, we used ELISA to analyse the level of secreted
cystatin F in the AD plasma with expanded cases, showing that cystatin F is significantly higher than that inage-matched controls (Fig. 3C), in accordance withthe changes in its transcript levels. As the disease progressed, plasma cystatin F levels progressively increased
depending on the AD stages (Fig. 3D). Furthermore, we
examined the correlation between the cystatin F dimer
concentration and the plasma Aβ40 or Aβ42 concentration. The results showed that the cystatin F dimer concentration was significantly positively correlated with theAβ40 or Aβ42 concentration (Fig. 3E, F). These findings
indicated that cystatin F dimer levels were correlatedwith Aβ levels. We also attempted to analyse the relationship between the cystatin F dimer concentration andthe clinical manifestations of AD. The MMSE, MoCA,RAVLT-I, RAVLT-D, and CDR are important tools thatprovide complete and accurate information about mental
health and cognitive status [40, 52]. Correlation analysis revealed that the plasma cystatin F concentration inpatients with AD was significantly negatively correlatedwith the MMSE, MoCA, RAVLT-I, and RAVLT-D scoresand positively correlated with the CDR (Fig. 3G-K). These
results implied that high levels of cystatin F dimers in theplasma of patients with AD are associated with Aβ levels,
the severity of AD, and the clinical features of AD.
Extracellular cystatin F dimer inhibits Aβ phagocytosis
by monocytic cells

Given that monocytes/macrophages are crucial phagocytic cells in the innate immune system, we first investigated whether high levels of the cystatin F dimer impact
the internalization of Aβ by monocytes. By the additionof purified cystatin F dimer protein to cultured monocytes to mimic the high concentration of cystatin F in
the plasma of AD patients, we found that the uptake ofAβ42-AlexaFluor 647 by the monocytic cell line THP-1(See figure on next page.)
Fig. 2 Monocyte-specific overexpression of human cystatin F exacerbated cognitive impairment in APP/PS1 transgenic mice. A Constructionof monocyte-specific overexpression of human cystatin F under the control of the human CD68 promoter. B Schematic for generating APP/
PS1/Hmo-cys ­F+ mice, behavioural tests, and pathological analysis. C Identification of transgenic mice by PCR analysis of the genomic DNA. D
Transcript levels of human cystatin F in the monocytes of 6-month-old WT (n = 6), Hmo-cys ­F+ (n = 6), APP/PS1 (n = 6), and APP/PS1/Hmo-cys ­F+mice (n = 6). E Immunofluorescence and statistical analysis of Aβ plaques in brain sections from transgenic mice. Scale bar: 1000 μm F, G ELISA
analysis for soluble F Aβ40 and G Aβ42 levels in brains extracted with RIPA buffer. H, I ELISA analysis for insoluble H Aβ40 and I Aβ42 levels in brains
extracted with SDS buffer. J, K MWM analysis showing the latency (s), L number of target crosses, and M mean speed (cm/s) in the invisible platform
test. N Representative images of the track plots in the MWM test. O, P ELISA analysis of O Aβ40 and P Aβ42 levels in the plasma of mice. All the micein the behavioural test and Aβ analysis were 12 months old and included WT (n = 6), Hmo-cys ­F+ (n = 7), APP/PS1 (n = 7), and APP/PS1/Hmo-cys­F+ mice (n = 8). The data are presented as the means ± SD and were analysed using Student’s t test and one-way ANOVA. *p < 0.05, **p < 0.01,***
p < 0.001, ****p < 0.0001

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Li et al. Journal of Neuroinflammation(2024) 21:125

gradually decreased with increasing concentrations of thecystatin F dimer via FCM analysis (Fig. 4A). The ELISA
data showed that the intracellular Aβ42 levels decreased
in a dose-dependent manner as the cystatin F dimer concentration increased (Fig. 4B). Additionally, we observed
less Aβ42-Alexa Fluor 555 in THP-1 cells pretreatedwith 250 ng/mL cystatin F dimer protein by LSCM
(Fig. 4C). Notably, when the substrates were changed tofluorescence-labelled transferrin or dextran, monocytes
exhibited similar levels of phagocytosis in the presence
and absence of cystatin F (sFig. 3A and B), indicatingthat cystatin F inhibits Aβ phagocytosis by monocyteswith a certain specificity. The process of internalizationby phagocytic cells is known to be actin dependent [53].
We further selected cytochalasin D and EIPA, which are
specific inhibitors of phagocytosis and macropinocytosis, respectively, to investigate the effect of the cystatin F
dimer on intracellular phagocytic signalling [54, 55]. As
shown in Fig. 4D, the two inhibitors effectively hindered
the internalization of Aβ by monocytes; moreover, thesuppressive effect was apparently reversed specificallyby the cystatin F dimer protein, whereas cystatin F failed
to reverse the inhibition of Aβ internalization as determined by the amount of fluorescently labelled dextran(sFig. 3C).
The recruitment of exogenous substances by monocytes is a prerequisite for phagocytosis. LSCM showed asignificant reduction in Aβ42-Alexa Fluor 555 adhesion
to the monocyte membrane when the cells were incubated in advance with the cystatin F dimer at 250 ng/mL(Fig. 4E). In addition, we used total TIRFM to observe
the enrichment of Aβ42-Alexa Fluor 555 on the cell
membrane of monocytes. The images showed that lessAβ42-Alexa Fluor 555 adhered to the surface of monocytes which were pretreated with purified cystatin F
dimer protein (Fig. 4F, G). Moreover, co-incubating thesubstrate with cells at a lower temperature effectively
prevented its internalization, showing that the cystatin
F dimer was able to markedly inhibit the adhesion of Aβ

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to monocytes at 0 °C, as determined by FCM and ELISA
analysis (Fig. 4H, I). These results demonstrated that thecystatin F dimer blocks monocyte phagocytosis by hindering the binding of Aβ to the monocyte membrane.
Cystatin F dimer physically interacts with Aβ to inhibit its
internalization by monocytes

High concentrations of the cystatin F dimer present
with Aβ in the circulation and inhibit the internalization of Aβ by monocytes; therefore, we first investigatedwhether there is a potential relationship between them.
Cystatin F shares a high homology with cystatin C, which
belongs to the same family and can interact with Aβ [56].
We explored the potential associations, assuming thatdimeric cystatin F binds to Aβ. A binding assay that canestimate the binding capacity of the two proteins showedthat the cystatin F dimer protein directly binds to Aβ40
and Aβ42 in a dose-dependent manner (Fig. 5A, B). BothGST and His pull-down assays further demonstrated astrong interaction between the cystatin F dimer and Aβ(Fig. 5C, D). Furthermore, we used BiFC to investigate
how the cystatin F dimer interacts with Aβ in vitro. Aftertagging the two proteins at their N-terminal and C-terminal fragments of fluorescent Venus protein (Fig. 5E),
the cystatin F dimer-VN protein and Aβ-VC protein
were expressed in 293T cells and purified with a nickel
agarose column. When the purified cystatin F dimerVN protein was incubated with the Aβ-VC protein, thefluorescence intensity was measured at 529 nm, and theresults indicated that the cystatin F dimer is capable ofbinding to Aβ (Fig. 5F). We further investigated whether
the interaction between the cystatin F dimer and Aβ led
to the inhibition of the recruitment and internalization ofAβ. We predicted the amino acids that interact with thecystatin F dimer and Aβ42, and three potential binding
mechanisms were identified by the Cluspro1, HDOCK2,
and MOE3 methods (Fig. 5G and sTable 3). The predicted
amino acids on the cystatin F dimer that are potentially capable of interacting with Aβ were mutated to(See figure on next page.)
Fig. 3 Identification of the expression and structure of the cystatin F protein in the plasma of AD patients and analysis of its correlation with AD
clinical manifestations. A Western blot analysis of the expression and structure of the cystatin F protein in plasma from patients with AD (n = 6)
and controls (n = 6) by using a nondenaturing gel. The total protein in the plasma was stained with CBB as an internal control. B The structureof cystatin F in human plasma was determined by western blot assay, and DTT was added to the sample to reduce the dimers to the monomers.
C ELISA analysis of secreted cystatin F levels in plasma from patients with AD (n = 45) and controls (n = 40). D ELISA analysis of secreted cystatin
F in plasma according to the AD stage from mild (n = 15) to moderate (n = 15) to severe dementia (n = 15). E, F Correlations of the cystatin F levelwith the Aβ40 and Aβ42 levels in the plasma of patients with AD by Pearson correlation coefficient analysis. G-K Correlations of cystatin F levels
with MMSE, MoCA, RAVLT-I, RAVLT-D and CDR scores by Pearson correlation coefficient analysis. The data are presented as the means ± SD and wereanalysed using Student’s t test and one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001

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Li et al. Journal of Neuroinflammation(2024) 21:125

alanine, namely, cystatin F (KSRKKKWWRK), cystatin
F (RTSTK) and cystatin F (YNR). GST pull-down assays
revealed that cystatin F (RTSTK) had a weaker ability tobind to Aβ42 than did cystatin F (KSRKKKWWRK) andcystatin F (YNR) (Fig. 5H–J), indicating that the Thr41,
Ser73, Arg77, Lys119, and Thr121 residues in cystatin F
form hydrogen bonds and that salt bridges with the Asp7,
Tyr10, His14, Gly33 and Leu34 residues in Aβ42 are
involved in these interactions (sFig. 3). Moreover, compared with the cystatin F dimer protein, the cystatin F(RTSTK) protein or supernatant of 293T cells transfectedwith the cystatin F (RTSTK) plasmid restored Aβ uptake
according to FCM and ELISA (Fig. 5K, L and sFig. 4A).
In addition, we observed that monocytes pretreated withthe cystatin F (RTSTK) mutant protein had an enhanced
ability to recruit Aβ compared to monocytes pretreatedwith the wild type (WT) cystatin F dimer protein through
TIRFM and LSCM (Fig. 5M and sFig. 4B). In parallel,
the FCM analysis results showed that the adhesion of
Aβ to monocytes that were pretreated with the cystatin
F (RTSTK) protein was significantly improved (Fig. 5N).
These results implied that the complex formed by theinteraction of the cystatin F dimer and Aβ extracellularlyis the major contributor to the failure of monocytes toeffectively recruit and internalize Aβ.
Tail vein injection of recombinant cystatin F dimers
aggravates Aβ deposition and cognitive impairment
in 5XFAD mice

Due to the increased expression of the cystatin F dimerin a collection of patients with sporadic AD, we further
explored whether cystatin F is associated with mutationsin AD-related genes. We detected the expression of cystatin F in 5XFAD transgenic mice, which express human
APP and PSEN1 transgenes with a total of five AD-linked
mutations, namely, the Swedish (K670N/M671L), Florida(I716V), and London (V717I) mutations in APP and the
M146L and L286V mutations in PSEN1 [57]. As shown

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in sFig. 5A, the expression of the cystatin F protein in theplasma of 5XFAD transgenic mice did not significantly
increase compared with that in the plasma of control
mice. Then, the 5XFAD transgenic mice, which exhibited many AD-related phenotypes at a relatively earlystage, received a high dose of purified mouse cystatin
F dimer via the tail vein to disperse into the peripheral
blood (Fig. 6A–C). We first assessed Aβ levels from thebrain to the periphery. In the brain, Aβ plaque coverage within the cortex and hippocampus significantly
increased (Fig. 6D), and the levels of Aβ40 and Aβ42
markedly increased in 5XFAD-cystatin F dimer mice
(Fig. 6E, F). The ELISA results showed that the levels ofsoluble Aβ40 and Aβ42 was increasing in the plasma of
5XFAD-cystatin F dimer mice compared with the control
mice (Fig. 6G, H). Next, we investigated whether bloodborne cystatin F dimers affect central Aβ clearance in thebrain. Through the detection of His tag-fused recombinant cystatin F proteins in brain sections, we found thatcirculating high-level cystatin F failed to diffuse into
the brain (sFig. 5B) and that microglia were not affectedby increased peripheral levels of cystatin F in 5XFAD
mice, either morphologically or in terms of numbers(sFig. 5C). Additionally, we used a human brain microvascular endothelial cell (HBMEC) monolayer cultured
on a Transwell insert by adding Aβ to the lower chamber to mimic the AD blood brain barrier model in vitro[58]. We found that monocyte transendothelial migration
was not altered by elevated levels of the cystatin F dimer(sFig. 5D and E). These results suggested that high levelsof the cystatin F dimer influence Aβ metabolism mainlyin the periphery.
Furthermore, various behavioural tests were carriedout to assess the effect of the cystatin F dimer protein
on learning and memory in 5XFAD mice. The MWM
results showed that 5XFAD-cystatin F dimer mice exhibited longer escape latencies during the training trials andfewer platform crossings but no significant alterations in(See figure on next page.)
Fig. 4 Cystatin F dimers inhibited the uptake of Aβ by monocytes. A-C THP-1 cells were pretreated with cystatin F dimer protein for 30 min,and 1 μg/mL soluble Aβ42-Alexa Fluor 647, or Aβ42, or Aβ42-Alexa Fluor 555 were added for an additional 30 min. Then, the cells were subjected
to A FCM, B ELISA and C LSCM analysis. D THP-1 cells were pretreated with 5 μg/mL cytochalasin D for 30 min, 40 μM EIPA for 1 h, and 250 ng/
mL cystatin F for 30 min. Then, cells were incubated with 1 μg/mL soluble Aβ42-Alexa Fluor 647 for 30 min and analysed by FCM method. E THP-1
cells were pretreated with 250 ng/mL cystatin F, and 1 μg/mL soluble Aβ42-Alexa Fluor 555 was added, then cells were incubated for 30 min
at 0 °C. Then, the cells were observed by LSCM. F, G Schematic diagram depicting the use of TIRFM for imaging membrane-localized proteins.
THP-1 cells were pretreated with 250 ng/mL cystatin F dimer for 30 min, and then 1 μg/mL soluble Aβ42-Alexa Fluor 555 was added and incubatedfor 30 min at 0 °C. Cells were observed by TIRFM. H, I THP-1 cells were pretreated with 250 ng/mL cystatin F, and 1 μg/mL soluble Aβ42-Alexa Fluor
647 or Aβ42 was added, then cells were incubated for 30 min at 0 °C. Then, the cells were subjected to H FCM and I ELISA. The data are presented
as the means ± SD and were analysed using Student’s t test and one-way ANOVA. **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 5 μm

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Li et al. Journal of Neuroinflammation(2024) 21:125

swimming speed (Fig. 6I–M). In the NOR test, 5XFADcystatin F dimer mice spent less time exploring new
objects, as evidenced by a significant decrease in the recognition index (Fig. 6N, O). Compared with those of thecontrols, the time, ambulation, and number of visits inthe novel arms were obviously decreased in the 5XFADcystatin F mice during the Y maze tests (sFig. 5F–I).
These results indicated that a high plasma cystatin F
dimer level could rapidly exacerbate Aβ deposition and amemory deficit in 5XFAD mice as a risk factor.

Discussion
The mechanism underlying the dysfunctional peripheral
clearance of Aβ by monocytes derived from AD patientsis unclear. Herein, we demonstrated that aberrantly elevated expression of the cystatin F dimer released by AD
monocytes is a determinant of dysfunctional Aβ clearance in the periphery and contributes to AD pathology.
Monocytes and macrophages are capable of physiologically removing circulating Aβ that originates in thebrain through multiple means. Similar to glial cells in thebrain, monocytes/macrophages in the periphery express
phagocytic receptors for Aβ and produce Aβ-degrading
enzymes to recognize and degrade Aβ [59, 60]. Mounting evidence suggests that gene expression changes inperipheral immune cells can affect brain function andanimal behaviour [61]. We demonstrated that cystatin
F expression was specifically elevated in the monocytesof sporadic AD patients and that in vivo, human cystatin F aggravated Aβ deposition in the brain and cognitive
impairment in APP/PS1 mice. Cystatin F was first identified in samples from AD patients by Keren-Shaul H et al.
[62] and was reported to be significantly upregulated in
microglia as an amyloid plaque’s indicator [63]. Cystatin
F exhibited similarly elevated expression and functions

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in contributing AD development in both peripheral
and central innate immune cells. This may be a resultof meningeal macrophages penetrating the brain during embryonic development to form microglia, and evenbrain parenchymal microglia could also be derived fromblood monocytes, which are regarded as the counterpartsof microglia in the periphery [64, 65]. Our findings verified the importance of defects in peripheral monocytes
in AD progression and that cystatin F may be a potential
risk factor.
Bone marrow-derived monocytes have been studied
often in AD for their ability to infiltrate the brain andengage in the phagocytosis of Aβ [66]. Since cystatin F isunable to cross the BBB and has no impact on resident
microglia and transendothelial migration of monocytes,
it may play a major role in Aβ clearance in the periphery.
We determined that cystatin F was released as a dimer
into the plasma. Additionally, the cystatin F dimer specifically impaired Aβ internalization by hindering its adhesion to the surface of monocytes. This finding not only
confirms that abnormal levels of cystatin F in plasma candirectly affect the phagocytosis of Aβ by monocytes but
also challenges the previous view that cystatin F is inactive in its dimeric form until it is reduced into monomers[67].
Cystatin F belongs to the type II cystatin gene family,
which shares approximately 38% homology with its family member cystatin C [68]. The role that Cystatin C plays
in AD progression is controversial, however Cystatin C
has been found to inhibit the expression of cathepsin B todegrade Aβ [69]. Moreover, cystatin C can bind to Aβ to
inhibit further oligomerization and fibrosis of Aβ to protect against neuronal cell death and limit the course ofAD [70, 71]. Although cystatin F is especially expressedin the microglia around Aβ plaques [72] and is regarded(See figure on next page.)
Fig. 5 Cystatin F dimers physically interact with Aβ to inhibit the internalization of Aβ by monocytes. A Schematic of the binding assay. B Plates
were coated with 4 ng/μL dissolved Aβ40, Aβ42, Aβ40-1, and Aβ42-1 for 16 h. Recombinant cystatin F dimers were added to the wells for 3 h.
The primary antibody for cystatin F and the secondary antibody coupled with HRP were used to generate a colour reaction, and the absorbancewas measured at 450 nm. C, D The GST pull-down assay and His pull-down assay were used to analyse the interaction between the cystatin F
dimer and Aβ. E Schematic diagram depicting the BiFC assay for detecting the interaction between the cystatin F dimer and Aβ. F The BiFC assay
was carried out as described in the Materials and Methods section. Then, 200 ng/mL cystatin F dimer-VN and 200 ng/mL Aβ-VC protein wereincubated at 37 °C for 2 h, and the fluorescence intensity was assessed at 529 nm. G Prediction of the amino acid interactions between cystatin
F dimer and Aβ. H-J The potential amino acids on cystatin F that interacted with Aβ were mutated to alanine. The interaction was determinedby a GST pull-down assay. K–L THP-1 cells were pretreated with 250 ng/mL cystatin F dimer protein or cystatin F (RTSTK) mutant protein for 30 min,
1 μg/mL Aβ42-Alexa Fluor 647 or Aβ42 was added for an additional 30 min, and the cells were subjected to K FCM and L ELISA analysis. M, N
THP-1 cells were pretreated with 250 ng/mL cystatin F dimer protein or cystatin F (RTSTK) mutant protein for 30 min, and 1 μg/ml Aβ42-Alexa Fluor
555 or Aβ42-Alexa Fluor 647 were added for an additional 30 min, and the cells were observed by M TIRFM and subjected to N FCM. The data arepresented as the means ± SD and were analysed using Student’s t test and one-way ANOVA. **p < 0.01, ***p < 0.001. Scale bar: 5 μm

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Li et al. Journal of Neuroinflammation(2024) 21:125

as a sensitive indicator of Aβ plaques in AD [31], thelink between cystatin F to Aβ has not been explored.
We found that the cystatin F dimer is able to physicallyinteract with Aβ through specific amino acids. Amongthese amino acids, arginine and lysine, which are locatedat positions 77 and 119, respectively, are also found in
the homologous region of cystatin F and cystatin C. The
direct interaction between the cystatin F dimer and Aβ
is the dominant force that inhibits the recognition andinternalization of Aβ by monocytes, as weakening theinteraction by destruction of the interacting amino acids
restored the uptake of Aβ by monocytes. Several secreted
proteins are able to alter the metabolism of Aβ by binding
to Aβ in the extracellular fluid. For instance, β2M markedly accelerated Aβ42 aggregation and oligomer formation by coaggregation with Aβ [73]. Whether the bindingof cystatin F to Aβ results in structural changes that affectAβ recognition by pattern receptors on monocyte membranes is worth exploring in depth.
Sporadic AD is a progressive disorder that typicallystarts in midlife, decades before symptom onset. Aβ
accumulation precedes the onset of other AD pathologies and a decrease in cognitive impairment [2]. Strategies targeting Aβ are thus promising disease-modifying
approaches for the treatment and prevention of AD [74].
Cystatin F dimers especially rapidly exacerbated the deposition of Aβ in the brain and cognitive deficits in 5XFAD
transgenic mice, suggesting that this protein is a promising target in peripheral blood. An important reason
for the uncontrollability of AD progression is the lack ofearly noninvasive diagnostic biomarkers. Considerable
research has focused on changes in certain signature
proteins of AD in the plasma because these proteins areeasily accessible and noninvasive [75, 76]. The increase in
the cystatin F dimer level in plasma from patients with
AD patients was closely correlated with the Aβ level andclinical mental score of patients with AD, suggesting thatthe cystatin F dimer level in plasma from patients withAD might be useful for assessing AD severity.

Page 16 of 20

There are several limitations to this study. First,
the interaction of the cystatin F dimer with Aβ likely
occurs through multiple amino acids; thus, it is difficult to use genetic or pharmacological approaches to
disrupt this interaction to enhance the recognitionand phagocytosis of Aβ by monocytes. Second, sexdifferences exist in clinical AD, and preclinical model
animals also exhibit sexual dimorphisms in AD-likebehaviours. In particular, Daniels MJD et al. recently
reported that cystatin F plays a sexually dimorphic
role in regulating microglia in Cst7-deficient AD miceand that the microglia of female mice have a greaterAβ burden in vivo [77]. To avoid the potential confounding effects of sex, only male mice were used inthis study. Thus, our findings in male mice may not
be completely recapitulated in female mice. Finally,
although cystatin F may be a potential target, reducing
or eliminating cystatin F from the periphery is a challenge for the future.

Conclusions
Our study revealed the pathophysiological importance of elevated circulatory cystatin F expression in
the periphery of AD patients. Specifically, we showedthat cystatin F expression is increased in the peripheral monocytes of AD patients, exacerbating Aβ deposition in the brain and the pathogenesis of AD. Most
interestingly, cystatin F in the plasma of AD patients
exists as a dimer and is closely correlated with theseverity and clinical manifestations of AD. Moreover,
we noted that the cystatin F dimer blocks the phagocytosis of Aβ by monocytes by interacting with Aβand, in vivo, aggravates Aβ deposition and cognitive
dysfunction in AD model mice. Overall, our findings
highlight that the cystatin F dimer plays a crucial role
in regulating Aβ metabolism in the peripheral clearance pathway, providing us with a potential biomarker
for diagnosis and a potential target for therapeutic
treatment.

(See figure on next page.)
Fig. 6 High-level cystatin F dimers in plasma rapidly aggravate cognitive impairment in 5xFAD transgenic mice. A Schematic of tail vein injectionof 5xFAD mice, behavioural tests, and pathological analysis. B The purified murine cystatin F dimer was identified by western blot analysis.
C The level of cystatin F in the plasma of the mice after tail vein injection of purified protein which was detected by ELISA analysis (n = 8). D
Immunofluorescence and statistical analysis of Aβ plaques in brain section from transgenic mice that received a tail vein injection. Scale bar:
500 μm E, F ELISA method for measuring Aβ40 and Aβ42 levels in the brain of the mice. G, H ELISA method for measuring Aβ40 and Aβ42 levelsin the plasma of the mice. I–L MWM analysis of the latency (s), number of target crosses, and mean speed (cm/s) in the invisible platform testof the mice. M Representative images of the track plots in the MWM test. N The motion traces of the NOR test were displayed. O The recognitionindex was calculated as described in Material and Methods. The data are presented as the means ± SD and were analysed using Student’s t test.
*p < 0.05, **p < 0.01, ***p < 0.001

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Li et al. Journal of Neuroinflammation(2024) 21:125

Abbreviations
AD	Alzheimer’s disease
APP	Amyloid precursor protein
AUC​	Area under the curve
BiFC	Bimolecular fluorescence complementation
CBB	Coomassie brilliant blue
CDR	Clinical dementia rating
DAM	Disease-associated microglia
DAPI	4′,6-Diamidino-2-phenylindole
DiOC18(3)	3,3′-Dioctadecyloxacarbocyanine perchlorate
DTT	Dithiothreitol
ELISA	Enzyme-linked immunosorbent assay
FC	Fold change
FCM	Flow cytometry
GAPDH	Glyceraldehyde-3-phosphate dehydrogenase
GO	Gene Ontology
GSEA	Gene set enrichment analysis
HBMEC	Human brain microvascular endothelial cell
Hmo-cys ­F+	
Human cystatin F under the control of thehuman CD68 promoter
HRP	Horse radish peroxidase
LSCM	Laser scanning confocal microscope
MMSE	Minimum mental state examination
MoCA	Montreal cognitive assessment
MW	Molecular weight
MWM	Morris water maze
NINCDS-ADRDA	The National Institute of Neurological and Communica‑tion Disorders and the Stroke and Alzheimer’s Disease andRelated Disorders Association
NOR	Novel object recognition
ORF	Open reading frame
PBS	Phosphate buffered saline
PEI	Polyethylenimine
PFA	Paraformaldehyde
RAVLT	Rey auditory verbal learning test
ROC	Receiver operating characteristic
RT-qPCR	Real-time polymerase chain reaction
TIRFM	Total internal reflection fluorescence microscopy
WT	Wild type

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12974-​024-​03119-2.
Additional file 1. Supplementary figures and tables.s
Acknowledgements
The authors are thankful to Dr. Chao Wang (Institute of Brain Science and Brain
Disease, Chongqing Medical University) for providing the 5xFAD transgenic
mice.
Author contributions
QL, BL, KJW, MYL, and ZL designed and performed the experiments andanalysed the data. LL and LYW diagnosed the AD patients and collected
the clinical samples. YD, WDZ and YHC commented on the manuscript. KZ
conceptualized the study, supervised the experiments, interpreted the results,
acquired funding, and prepared the manuscript.
Funding
This work is supported by the National Natural Science Foundation of China(81571046), the Key Project of The Educational Department of Liaoning
Province (LJKZ0755), the Project of the Department of Science & Technology
of Liaoning Province (2023JH2/20200116), the Shenyang Young and Middleaged Innovative Talents Support Program (RC210240), and the Shenyang
Science and Technology Project (21-173-9-22).

Page 18 of 20

Availability of data and materials
All data generated or analysed during this study are included in this publishedarticle and its supplementary information files. The datasets produced during
the current study are also available from the corresponding author upon
reasonable request.

Declarations
Ethics approval and consent to participate
All the animal experimental procedures were approved by the Animal Experi‑mentation Ethics Committee of China Medical University (CMU20240091). All
participants (or their legal guardians) provided written informed consent.
Consent for publication
All authors agreed to publish this study.
Competing interests
The authors declare no competing interests.
Author details
1
Department of Developmental Cell Biology, Key Laboratory of Cell Biology,,
Ministry of Public Health, China Medical University, 77 Puhe Road, Shen‑yang 110122, China. 2 Department of Laboratory Medicine, Shengjing Hospitalof China Medical University, Shenyang 110004, China. 3 Department of Neurol‑ogy, Xuanwu Hospital, Capital Medical University, Beijing 100053, China.
4
Department of Neurology, Shenyang Fifth People Hospital, Shenyang 110023,
China. 5 Department of Environmental Health, School of Public Health, China
Medical University, Shenyang 110122, China.
Received: 4 March 2024 Accepted: 1 May 2024

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