Song et al. Journal of Neuroinflammation(2024) 21:167
https://doi.org/10.1186/s12974-024-03162-z

Journal of Neuroinflammation

Open Access

RESEARCH

Dysregulated expression of miR-140 and miR122 compromised microglial chemotaxis
and led to reduced restriction of AD
pathology
Chao Song1†, Shufang Li1†, Yingren Mai2†, Linpeng Li1, Guoku Dai1, Yuan Zhou1, Xiaosheng Liang1,3,
Olivia Meilan Zou1, Ya Wang1, Libing Zhou3*, Jun Liu2* and Yi Zou1*
Abstract
Background Deposition of amyloid β, which is produced by amyloidogenic cleavage of APP by β- and γ-secretase,is one of the primary hallmarks of AD pathology. APP can also be processed by α- and γ-secretase sequentially,
to generate sAPPα, which has been shown to be neuroprotective by promoting neurite outgrowth and neuronalsurvival, etc.
Methods The global expression profiles of miRNA in blood plasma samples taken from 11 AD patients as well asfrom 14 age and sex matched cognitively normal volunteers were analyzed using miRNA-seq. Then, overexpressed
miR-140 and miR-122 both in vivo and in vitro, and knock-down of the endogenous expression of miR-140 and miR122 in vitro. Used a combination of techniques, including molecular biology, immunohistochemistry, to detect the
impact of miRNAs on AD pathology.
Results In this study, we identified that two miRNAs, miR-140-3p and miR-122-5p, both targeting ADAM10, the main
α-secretase in CNS, were upregulated in the blood plasma of AD patients. Overexpression of these two miRNAs inmouse brains induced cognitive decline in wild type C57BL/6J mice as well as exacerbated dyscognition in APP/PS1
mice. Although significant changes in APP and total Aβ were not detected, significantly downregulated ADAM10
and its non-amyloidogenic product, sAPPα, were observed in the mouse brains overexpressing miR-140/miR-122.
Immunohistology analysis revealed increased neurite dystrophy that correlated with the reduced microglial
chemotaxis in the hippocampi of these mice, independent of the other two ADAM10 substrates (neuronal CX3CL1†

Chao Song, Shufang Li and Yingren Mai contributed equally to thiswork.
*Correspondence:
Libing Zhou
tlibingzh@jnu.edu.cn
Jun Liu
liujun@gzhmu.edu.cn
Yi Zoutyizou@jnu.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 the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Song et al. Journal of Neuroinflammation(2024) 21:167

Page 2 of 19

and microglial TREM2) that were involved in regulating the microglial immunoactivity. Further in vitro analysis
demonstrated that both the reduced neuritic outgrowth of mouse embryonic neuronal cells overexpressing miR140/miR-122 and the reduced Aβ phagocytosis in microglia cells co-cultured with HT22 cells overexpressing miR140/miR-122 could be rescued by overexpressing the specific inhibitory sequence of miR-140/miR-122 TuD as well as
by addition of sAPPα, rendering these miRNAs as potential therapeutic targets.
Conclusions Our results suggested that neuroprotective sAPPα was a key player in the neuropathologicalprogression induced by dysregulated expression of miR-140 and miR-122. Targeting these miRNAs might serve as apromising therapeutic strategy in AD treatment.
Keywords Alzheimer’s disease, Microglial, Neuroinflammation

Background
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that causes the most common form ofdementia in the elderly [1]. The underlining pathologicalevents in brains are characterized by the senile plaqueand neurofibrillary tangles made by accumulated betaamyloid (Aβ) and hyperphosphorylated tau protein,respectively [2]. Aβ proteins were produced by amyloidogenic proteolysis of amyloid precursor proteins (APP),
which are transmembrane proteins displaying G protein-dependent receptor activity [3]. APP can be cleavedby α- or β-secretase to produce soluble APPα (sAPPα)or APPβ, the C-terminal fragment of the latter further
cleaved by γ-secretase to release small Aβ peptides ranging from 38 to 43 amino acids [4]. The fragment Aβ42
tend to form self-aggregates, which are believed to beneurotoxic and are the predominant forms found in AD
amyloid plaques [5]. It is currently proposed that soluble
Aβ oligomers, rather than monomers or fibrils, are thecytotoxic species to induce neuroinflammation, oxidative stress, synaptic dysfunction, and eventually neuronalloss [6, 7]. The majority of brain-derived Aβ, up to 60%, iscleared in the periphery via receptor-mediated transport
across BBB as well as via interstitial pathway into CSF[8]. The Aβ peptides can also be up taken by phagocytosis of microglia and astrocytes, which also degrade Aβ by
secreting a number of proteases [9]. However, Aβ peptide
also displayed neuroprotective function at low concentrations [10, 11]. Therefore, the life-long proper Aβ production is dependent on the precise adjustment of Aβ
generation and clearance, both of which are fundamental
therapeutic strategies regarding AD treatment [12–14].
Unlike the amyloidogenic peptides, sAPPα appeared
to be neuroprotective, although the detailed downstream pathway remained unclear. It was shown to promote neurite outgrowth, enhance synaptogenesis, anddisplayed neurotrophic effect to prolong neuronal survival in deleterious environment [15, 16]. ADAM10, a
member of ADAM (a disintegrin and metalloprotease)family, is the main α-secretase that cleaves APP in brain[17, 18]. The expression of ADAM10 is intricately regulated and among them, the transcriptional regulation by

all-trans retinoic acid (atRA) has been well studied [19].
RA-induced ADAM10 expression has been shown to bebeneficial in AD models both in vitro and in vivo [20,21]. Diminishing ADAM10 activities, via mutations in
the functional domain of ADAM10 in AD mice, lead toincreased amyloidogenic processing of APP and significantly reduces neurogenesis [13].
Here we reported that two miRNAs, miR-140-3p
and miR-122-5p (referred to as miR-140 and miR-122
in this work), both targeting ADAM10, were upregulated in the blood plasma of AD patients as well as in
the brains of APP/PS1 mice. Overexpression of miR140 and miR-122 in mouse brains significantly downregulated ADAM10 and induced cognitive impairment
in wild type mice as well as exacerbated cognitive dysfunction in APP/PS1 mice. Treatment of atRA significantly improved cognition in mice overexpressing
miR-140 and miR-122 via upregulating the expression of
ADAM10. Reduced microgliosis and microglia activation were observed in the hippocampi of APP/PS1 miceoverexpressing miR-140/miR-122. These inert microglia cells displayed reduced chemotaxis and phagocytosis and thus exacerbated neuritic dystrophy in AD mice.
The neuritic dystrophy in cultured embryonic neuronal cells overexpressing miR-140/miR-122 and the inert
microglia co-cultured with neuronal cells overexpressing miR-140/miR-122 could be rescued by atRA treatment as well as by addition of sAPPα but not by blocking
with anti-Aβ. In addition, overexpression of the specificinhibitory miR-140/miR-122 TuD significantly enhanced
neurite outgrowth and microglial phagocytosis in vitro.
Taken together of the results in this study, we proposedthat reduced neuroprotective sAPPα played a key role
in regulating the neuropathological progression associated with dysregulated miR-140 and miR-122 expressionin AD mice. Targeting these miRNAs might serve as apotential therapeutic strategy in AD treatment.

Materials and methods
Reagents

DMEM, fetal bovine serum (FBS), and Lipofectamine™
2000 transfection reagents were purchased fromSong et al. Journal of Neuroinflammation(2024) 21:167

Invitrogen. 1,4-dithiothreitol (DTT), dimethyl sulfoxide(DMSO), Triton X-100, 4% paraformaldehyde, bovine
serum albumin, ethanol, Hoechst 33,342, RIPA buffer,phosphatase inhibitor cocktail, all-trans retinoic acid,corn oil, thioflavine S, penicillin, and streptomycin werefrom Sigma. Blue RangeTM prestained protein molecular marker and BCA Protein Assay Kit were from Thermo
Fisher Scientific. Recombinant Human Amyloid Precursor Protein alpha was purchased from Antibodies.com(Cambridge, UK). Anti-ADAM10 antibody (Cat. No.
ab124695, 1:5000) and anti-CX3CL1 antibody (Cat. No.
ab25088, 1:1000) were purchased from Abcam (Cambridge, UK). Anti-APP antibody (Cat.No.2045, 1:2000)
and was purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-6E10 antibody (Cat. No. 803014,
1:5000) was purchased from Biolegend (San Diego, CA,
USA). Anti-beta Amyloid antibody (MOAB-2, Cat. No.
13075, 1:500) was purchased from Novus Biologicals(Littleton, CO, USA). Anti-Iba1 antibody (Cat. No. 01919741, 1:1000) was purchased from FUJIFILM Wako
Pure Chemical Corporation (Osaka, Japan). Anti-TREM2
antibody (Cat.No.27599-1-AP, 1:1000) was purchasedfrom Proteintech (Chicago, IL). Anti-CX3CR1 antibody(Cat.No.38,481, 1:1000) was purchased from Signalway
Antibody (SAB, College Park, Maryland, USA). HRPconjugated Affinipure Goat Anti-Mouse IgG (H + L)
(Cat. No. SA00001-1, 1:3000), HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (Cat. No. SA00001-2;
1:3000) were purchased from Proteintech (Chicago, IL).
Alexa Fluor 647-conjugated Affinipure Goat Anti-Mouse
IgG (H + L) (Cat. No. 115-605-003, 1:1000), Cy3-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Rabbit IgG(H + L) (Cat. No. 111-166-144, 1:1000) was purchasedfrom Jackson ImmunoResearch Laboratories (WestGrove, PA, USA). MiR-140-3p and miR-122-5p mimics/
scramble RNA were synthesized by GenePharma (Shanghai, China). The miR-140-3p mimics and miR-122-5p
mimics were double-stranded (5ʹ-​U​A​C​C​A​C​A​G​G​G​U​A​G​
A​A​C​C​A​C​G​G-3ʹ and 5ʹ-​U​G​G​A​G​U​G​U​G​A​C​A​A​U​G​G​U​G​U​
U​U​G-3ʹ, respectively). The scramble RNAs was doublestranded RNA (5ʹ- ​U​U​C​U​C​C​G​A​A​C​G​U​G​U​C​A​C​G​U​T​T-3ʹ).
Participants and sample collection

The participants, aged between 60-90-year-old, wererecruited from the Department of Neurology, the Second
Affiliated Hospital of Guangzhou Medical University andthe study were approved by the ClinicalTrials.gov (ID:
NCT03653156). All patients gave written informed consent. All participants underwent the Mini-Mental State
Examination (MMSE) and those scored > = 27 was considered as cognitive normal controls. Participants witha MMSE score < = 26 went through additional tests of
cerebrospinal fluid Aβ and p-Tau. Diagnosis of AD wasPage 3 of 19

made in accordance with the 2018 NIA-AA Pathological Diagnostic Guidelines. Aβ42 < 550 pg/mL or Aβ42/
Aβ40 ≤ 0.05 (Amyloid-beta (1–40) High Sensitive ELISA
and Amyloid-beta (1–42) High Sensitive ELISA, Cat. No.
RE59781, Cat. No. RE59791, IBL International GmbH.,
Hamburg, Germany), p-Tau > 50 pg/mL (phosphoTAU
ELISA, Cat. No. 30,121,609), and t-Tau > 399 pg/mL(hTau total ELISA, Cat. No. RE59631) was considered
ATN positive, and these participants were included in
the study providing no presence of other brain diseases.
The final cohort consisted of 11 AD and 14 cognitive normal subjects. 2 mL peripheral blood from the cubital vein
were collected using EDTA anticoagulation tubes (Cat.
No.367863, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) in the early morning after refraining from eating and drinking for eight hours and wereallowed to sit for 30 min at room temperature. Plasma
samples were collected from the supernatant after centrifugation at 3,000 rpm for 10 min and stored in 100 µL
aliquots at -80℃ before use.
Animals

4-month-old male APP/PS1 mice and control C57BL/6
mice were purchased from The Experimental Animal
Center of Guangdong (Guangzhou, China) and bredin the Laboratory Animal Management Center of the
Jinan University in a pathogen free facility. All mice werehoused at temperature (21–25℃) under 12 h- light/
dark cycle with free access to water and food. All animal experiments were approved by the Institutional
Animal Care and Use Committee of Jinan University(IACUC-20230625-02). For atRA treatment, atRA was
dissolved in DMSO and diluted in corn oil to make thefinal concentration is 5% DMSO (v/v). atRA (20 mg/kg)
was applied by intraperitoneal injection three times per
week. The control mice were mock treated with vehicles.
Administration of atRA was initiated 24 h after the stereotactic injection and continued for 8 weeks. All animals
were sacrificed using CO2 asphyxiation before the tissue
samples were collected.
Stereotactic injection

Mice were anesthetized with 2% pentobarbital sodium(50 mg/kg) and placed in the stereotactic platform (Harvard Apparatus, Holliston, MA). AAV9-miR140-miR122eGFP or AAV9-Scr-eGFP (4 × 1010 genome copies ×2) were injected into both sides of the lateral ventricles(posterior: +0.3 mm; mediolateral: ± 1.0 mm, dorsal:-2.0 mm) at a rate of 0.4 µL/min using a micro-drive
arm of the micropump (KDS Legato™ 130 micro-pump,
KD Scientific Inc., Holliston, MA) attached to a Hamilton 5 µL syringe (87930 Hamilton, Reno, NV). The needle was held in place for 5 min to prevent leakage before

Song et al. Journal of Neuroinflammation(2024) 21:167

retraction after each injection. Mice were recovered in a
warming cabinet before returning to their home cage.
Behavioral assessment

Mice were brought to the testing room and allowed toacclimate for 1 h before assessment. A Y-maze apparatus
composed of three enclosed arms (35 cm long × 15 cm
high × 5 cm wide) oriented at 120° angles from eachother was used for the Y maze test to assess short-term
spatial working memory. Mice were introduced at theend of one of the arms and allowed for 8-min free exploration. A spontaneous alternation was defined as consecutive entries into three arms. Morris water maze (MWM)
test was performed using a circle pool with a diameter of
120 cm diameter and a depth of 60 cm filled with water atroom temperature (24℃). The pool was virtually divided
into four quadrants and the escape platform (10 cm
diameter) was placed in one of the quadrants submerged
1 cm under water level. The test started by placing themouse in the pool at different quadrants in each training
and the mouse were allowed to find the platform withina maximum trial time of 90 s. The trial was completed if
the mice reached the platform within that time and wasallowed to remain on the platform for 10 s before returning. If the mouse was unable to find the platform within
90 s, it was guided to the platform and allowed to stay for
10 s. The training was performed on 4 successive days
with two trials per day, with one hour gap between. The
probe trial was performed on the fifth day with the platform removed from the pool. Initiation started from thefarthest direction from the position of platform used during training. The time spent in the platform quadrant andthe number of crossing the platform location were calculated. The activities of the mice in all behavioral tests
were recorded using a video tracking camera and analyzed using ANY-maze software (Stoelting Co. USA).
miRNA sequencing data processing

Total RNAs from plasma samples were extractedusing mirVana miRNA Isolation Kit (Cat.No.AM1561,
Ambion, Austin, TX, US) following the manufacturer’s
instructions. The RNA concentrations were determinedby the absorbances at 260 nm using NanoDrop ND-2000
spectrophotometer (Thermo Fisher Scientific, Waltham,
MA, USA). Libraries for sequencing were prepared using
10 ng of total RNA using QIAseq miRNA Library Kit(Cat.No. 331502, Qiagen, Germantown, MD, USA). In
brief, adapters are ligated sequentially to the 3’ and 5’
ends of miRNAs, followed by universal cDNA synthesis,
cDNA cleanup, library amplification and library cleanup.
Constructed libraries were quantified and validated using
Qubit 2.0 Fluorometer (Life Technologies, Carlsbad,
CA, USA) and 2400 bioanalyzer (Agilent Technologies,
USA) system, and were then sequenced on an Illumina

Page 4 of 19

NovaSeq 6000 system using PE150 model (Shanghai
Biotechnology Corporation) at an average read depth of
20–35 million reads per sample.
Raw reads were processed with fastx (version 0.0.13,
http://hannonlab.cshl.edu/fastx_toolkit/) to remove
adaptors, low-quality reads, and reads shorter than 10 nt
to obtain clean reads. The clean reads (18–40 nts) werealigned to miRNAs in miRbase20.0 (http://www.mirbase.org/) using Bowtie2 software (version 2.4.4) [22, 23].
Read counts were normalized using the trimmed meanof M-values (TMM) method, and then referenced thesequencing depth to get values of transcripts per million(TPM) [24, 25]. In addition, for unannotated miRNAs,
we used miRCat pipeline in the UEA sRNA workbench(version 4.4) to evaluate potential novel miRNA candidates according to miRNA precursor hairpins [26].
Bioinformatic analysis

miRNAs were further filtered by expression level to
exclude low expression genes using the ‘filterByExpr’function and a count value > = 10 in over 50% of the samples in each group (n > 7 in AD group and n > 6 in control group) was used as cut-off. The gene counts werenormalized by the method of trimmed mean of M-values
using the ‘calcNormFactors’ function of edgeR. Principle component analysis of the normalized miRNA
expression data after filtering were performed using the‘prcomp’ function and the differentially expressed miRNAs (DEMs) between the controls and AD patients wasanalyzed using the ‘glmQLFit’ function of edgeR [27,28]. The fold-change of at least 2 with adjusted p-value(FDR) < 0.05 was used as the cutoffs. The heatmap ofDEMs was generated using ‘pheatmap’ package and therelation between groups was depicted by hierarchicallyclustered according to Pearson correlation and average
linkage. The volcano plot, which the x-axis represents thelog2-transformed fold change and the y-axis the log10transformed p-value was constructed by using ‘ggplot2’
package The above process is all implemented in R (version 4.2.1). Comprehensive Automatically Mined Database of Human Diseases (Malacards) was used to searchcognitive dysfunction related genes for a gene list. Online
analysis tools of TargetScan (version 8.0) and microTCDS (version 5.0) were used for miRNA target gene prediction. ‘VennDiagram’ package of R software (version
4.2.1) was employed to depict the intersections of target
genes of DEMs and cognitive dysfunction related genes.
The miRNA-mRNA regulatory network was visualized
by using Cytoscape (version3.9.1). Functional annotation of Gene Ontology (GO, Biological Process, Cellular
Component, Molecular Function) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment
analysis of cognitive dysfunction related miRNA targets
were performed using DAVID (v2022q2, https://david.

Song et al. Journal of Neuroinflammation(2024) 21:167

ncifcrf.gov/) based on total miRNA targets as a population background.
miRNA mimics, recombinant AAV vectors and miR-140miR-122 TuD expression vectors

Mimics of miR-140 and miR-122, and scramble miRNA
were purchased from GenePharma (Shanghai, China).
The sequences of miR-140-3p and miR-122-5p wereobtained from miRBase database (www.mirbase.org).
The sequence of the stem loop structure of these two
miRNAs (Table S4) were cloned into the expression plasmids GV412 (Genechem, Shanghai, China). The AAV
vector GV412 contains CMV promoter and encoding
eGFP. The 293T cells were transfected with the recombinant AAV vectors, pHelper and pAAV9 rep-cap using
Lipofectamine 2000. After transfection for 48 h, cells
were harvested and lysed by four freeze-thaw cycles in
dry ice and 37℃ water baths for collect the virus. The
lysed incubation with benzonase (Sigma Aldrich) at 37℃
for 30 min to concentrate virus. Then, the virus was purified by the CsCl gradient ultracentrifugation and were
further concentrated on Amicon-15 Centrifugal Filters(Merck Millipore, Burlington, MA, USA). AAV genome
copies were titrated using quantitative PCR (qPCR).
DNA sequences encoding tough decoy RNAs targeting
both miR-140-3p and miR-122-5p, designed according
to Haraguchi et al., were synthesized by Sangon Biotech(Shanghai, China) and were cloned into the NheI/HindIIIsites of pcDNA3.1 [29]. The miR-140-miR-122-TuD contains two tandem stem-loop structures, each of whichcontains either the mmu-miR-140-3p or mmu-miR122-5p binding sites flanked by an 18-bp long stem and a
26-bp stem-loop, respectively. A 4-nt long mismatch wasinserted into each of the miRNA-binding sites between
10 and 11 from the 3’-end to avoid complete paring. The
schematic representation and sequence of the miR-140miR-122-TuD is shown in Fig. 2. The sequences of all
constructs were verified by sequencing (Sangon Biotech,
Shanghai).
RNA extraction and quantitative real time-PCR (qRT-PCR)

For isolation of miRNAs from blood plasma, the miRNAs were isolated from 100 µL of blood plasma using themiRNeasy Serum/Plasma Kit (Cat.No. 217184, Qiagen,Germantown, MD, USA) according to the manufacturer’s protocols. 1 pmol synthetic Caenorhabditis elegans
miRNA cel-miR-39 was used as an internal standard andadded to each sample. The final RNA was eluted with
10 µL of nuclease-free water and 3.75 µL RNA of eachsample was reverse transcribe using the Mir-X miRNA
First-Strand Synthesis Kit (Cat.No. 638313, Takara,
Ohtsu, Japan). The primer pairs consist of a common
mRQ 3’ primer and one of the specific 5’ primers (hsamiR-140-3p: 5’-​A​A​G​T​T​G​C​A​T​A​C​C​A​C​A​G​G​G​T​A​G​A-3’,

Page 5 of 19

hsa-miR-122-5p: 5’-​A​A​C​A​C​G​C​T​G​G​A​G​T​G​T​G​A​C​A​A-3’,
cel-miR-39:
5’-​A​C​C​G​A​G​G​T​T​C​A​C​C​G​G​G​T​G​T​A​A-3’)
were used to amplify hsa-miR-140-3p, hsa-miR-122-5p
and cel-miR-39, respectively.
For RNA isolation from mouse tissues, the mouse hippocampal and cortical tissues were first homogenized
and the total RNA was extracted using Trizol-chloroform
and isopropanol. 1 µg of total RNA was reverse transcribe using specific stem-loop primer (miR-140-3p: 5’-​
G​T​C​G​T​A​T​C​C ​A​G​T​G ​C ​A​G ​G ​G​T​C​C​G​A​G ​G​T​A​T​T​C​G ​C ​A​C​T​G​G​A​T​A​C​G​A​C​C​C​G​T​G​G-3’, miR-122-5p: 5’-​G​T​C​G​T​
A​T​C​C​A​G​T​G​C​A​G​G​G​T​C​C​G​A​G​G​T​A​T​T​C​G​C​A​C​T​G​G​A​
T​A​C​G​A​C​C​A​A​A​C​A-3’, snRNA-U6: 5’-​C​G​C​T​T​C​A​C​G​A​
A​T​T​T​G​C​G​T​G​T​C​A​T-3’) to synthesize cDNAs using thePrimeScript RT reagent Kit with gDNA Eraser (Cat. No.
RR047A, Takara, Kusatsu, Japan). The primer pairs (5’-​A​
A​G​T​T​G​C​A​T​A​C​C​A​C​A​G​G​G​T​A​G​A-3’ and 5’-​C​A​G​T​G​C​
A​G​G​G​T​C​C​G​A​G​G​T-3’, 5’-​A​A​C​A​C​G​C​T​G​G​A​G​T​G​T​G​A​
C​A​A-3’ and 5’-​C​A​G​T​G​C​A​G​G​G​T​C​C​G​A​G​G​T-3’) wereused to amplify mmu-miR-140-3p and mmu-miR-122-5p,
respectively. The primer pairs (5’-​C​C​G​T​T​C​C​T​G​C​G​T​T​
C​T​T​A​T-3’ and 5’- ​A​C​T​G​G​T​T​G​C​C​C​T​T​G​G​T​A​G-3’, 5’-​A​T​C​T​G​A​C​C​T​T​C​C​T​G​C​C​C​T​C​C​A​C-3’ and 5’-​A​C​C​C​A​
A​G​A​G​C​A​C​A​C​A​C​G​A​C​A​T​T​C-3’) were used to amplify
Chi3l1 and H2-Ea cDNA. cDNA of Gapdh was amplified
using the primer pairs (5’-​G​G​C​A​A​A​T​T​C​A​A​C​G​G​C​A​C​
A​G​T​C​A​A​G-3’ and 5’-​T​C​G​C​T​C​C​T​G​G​A​A​G​A​T​G​G​T​G​A​
T​G​G-3’) and was used as the internal reference of gene
expression. Quantitative real-time PCR was performed
using TB Green Premix Ex TaqII (Tli RNaseH Plus) (Cat.
No. RR820A, Takara, Kusatsu, Japan) on an Applied Biosystems Quanstudio3 platform (Applied Biosystems,
Waltham, MA). The relative expression of genes was calculated using the 2−ΔΔCt formula and were normalizedusing internal controls of cel-miR-39, U6 and Gapdh,
respectively.
Western blot

The cells and mouse brain tissues were lysed in RIPA buffer (Sigma) containing PMSF and phosphatase inhibitor
cocktail. Protein concentration was determined using
BCA Protein Assay Kit (Thermo Fisher Scientific). 20 µg
proteins of each sample were separated on 10% SDSPAGE gel and were then transferred onto PVDF membranes (BioRad). The membranes were blocked with 5%
skim milk in 0.1% TBS-Tween 20 for 1.5 h under room
temperature and probed with primary antibodies overnight at 4℃. The membranes were washed three times
with 0.1% TBS-Tween 20 and probed with HRP labeledsecondary antibodies for 2 h at RT. The immunoblots
were visualized using Amersham Imager680 (GE Healthcare Bio-Sciences Corp, Piscataway, NJ) and the bands
intensities were quantified by densitometric analysis with
ImageJ software (ImageJ 1.8.0).

Song et al. Journal of Neuroinflammation(2024) 21:167

Page 6 of 19

Fig. 1 miRNA profiling of the blood plasma of AD patients and cognitively normal controls. (A). PCA analysis of the normalized expression values ofplasma miRNA, showing the separation between AD patients and controls on the first principal component. (B). Heat map showing the clustering of differentially expressed miRNAs. (C). Volcano plot displaying the fold change and p value of DEMs, with the labeling of all DEMs with |log2-fold change| >1
and FDR adjusted p < 0.05. (D). miRNA–mRNA regulatory network visualization for the enrichment analysis of DEMs. The size of miRNA nodes indicatedthe number of their target genes. Red circles represent DEMs and pink/green circles are their target genes, which green circles also are cognitive dysfunction related genes. (E) Gene Ontology biological process terms and Kyoto Encyclopedia of Genes and Genomes pathways in enrichment analysis of
DEMs targets. Dot size represents the fold enrichment. The color of each dot represents the category of ontology, and the X-axis represents the p-value.
(F). Quantitative RT-PCR validation of the expression of miR-140 and miR-122 in the plasma of AD patients and controls. The results were presented asmeans ± SD (n ≥ 9). (G-H). Quantitative RT-PCR data showing the expression of miR-140 (G) and miR-122 (H) in the cortex and the hippocampi of C57BL/6J
mice and the APP/PS1 mice, respectively. The results were presented as means ± SD (n ≥ 3). *p < 0.05, ** p < 0. 01. Statistical analyses were performed using
a two-tailed unpaired Student’s t-test

Quantification of Aβ plaques, number of microglia cells,
Iba1-positive areas, microglia coverage and dystrophic
neurites using immunohistochemistry

Mice were anesthetized with 2% pentobarbital sodium(50 mg/kg) and perfused with 0.9% saline. The mousebrain was removed in ice and fixed in 4% paraformaldehyde (PFA) overnight at 4℃, followed by graded sucrose
solutions (20%, 30% in PBS) at 4℃. Hemisphere brains
were embedded in O.C.T. compound and store at -80℃.
15-µm and 30-µm-thick serial coronal sections of hippocampus region were prepared with a cryostat microtome(CM1950, Leica). For immunolabeling of Aβ deposits,
15-µm-thick sections were washed with PBS and permeabilized with 0.1% Triton X-100 in PBS for 15 min at room

temperature (RT). After blocking with 10% goat serum
in PBS at RT for 1 h, the tissue sections were subjected
to overnight incubation at 4℃ with primary antibodies (MOAB-2, 1:1000, Cat. No. 13075, Novus Biologicals, Littleton, CO, USA and anti-Iba1, 1:1000, Cat. No.
016-20001, Wako Chemical, diluted in PBS + 1.5% goat
serum), respectively. The sections were then washed with
0.1% Triton X-100 in PBS 5 min for 3 times, followed byincubation with Alexa Fluor 647-conjugated AffinipureF(ab′ )₂ Goat Anti-Mouse IgG (1:1000, Cat. No. 115605-003, Jackson ImmunoResearch Laboratorie) andCy3-AffiniPure F(ab′ )₂ fragment Goat anti-Rabbit IgG(1:1000, Cat. No. 111-166-144, Jackson ImmunoResearchLaboratorie) for 1 h at room temperature, respectively.

Song et al. Journal of Neuroinflammation(2024) 21:167

Page 7 of 19

Fig. 2 Dysregulated expression of miR-140 and miR-122 affected sAPPα production via targeting ADAM10. (A). The specific inhibitory sequence of miR140-miR-122-TuD containing two tandem stem-loop structures was cloned into the NheI/HindIII sites of pcDNA3.1. (B). The sequences of miR-140-miR122-TuD, with each of the stem -loop contains two miRNA-binding sites flanked by an 18-bp long stem and a 26-bp stem-loop, respectively. A 4-nt longmismatch was inserted into each of the miRNA-binding sites between 10 and 11 from the 3’-end to avoid complete paring. (C). Quantitative real-time PCR
showing significantly upregulated or downregulated miR-140 and miR-122 expression levels in 293T cells transfected with mimics of miR-140 and miR-122
or pcDNA3.1-miR-140-miR-122-TuD. Untransfected cells and cells transfected with scramble miRNA and pcDNA3.1 were used as controls. The results were
presented as means ± SD (n = 5). *p < 0.05, ****p < 0.0001. Statistical analyses were performed using One-way ANOVA. (D-I). Cultured 293T cells (D-F) andHT22 cells (G-I) were transfected with: (1) vehicle only; (2) scramble miRNA; (3) mimics of miR-140; (4) mimics of miR-122; (5) mimics of miR-140 and miR122; (6) pcDNA3.1; (7) pcDNA3.1-miR-140-miR-122-TuD; (8) mimics of miR-140 and miR-122 + mock treatment; (9) mimics of miR-140 and miR-122 + 2 µM
atRA. The control cells were treated with vehicles. (D-F). Western blot analysis and densitometric quantification of the relative expression of ADAM10 (D),sAPPα (E) and APP (F) in the transfected 293T cells as indicated. The results were presented as means ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. Statistical analyses were performed using One-way ANOVA or two-tailed unpaired Student’s t-test. (G-I). Western blot analysis and densitometric quantification of
the relative expression of ADAM10 (G), sAPPα (H) and APP (I) in the transfected HT22 cells as indicated. The results were presented as means ± SD (n = 3).
*p < 0.05, **p < 0.01, ***p < 0.001. Statistical analyses were performed using One-way ANOVA or two-tailed unpaired Student’s t-test. (J). ELISA analysis of
the soluble Aβ42 monomer in the cultured HT22 cells transfected with scramble miRNA, mimics of miR-140 and miR-122, pcDNA3.1 and pcDNA3.1-miR140-miR-122-TuD. The results were presented as means ± SD (n = 3). *p < 0.05, statistical analyses were performed using One-way ANOVA

The nuclei were counterstained using Hoechst 33342(1:10000, Cat. No. 14533, Sigma-Aldrich) in Fluoromunt™
Aqueous Mounting Medium (Cat. No. F4680; SigmaAldrich) and coverslipped before imaging. The unilateralhippocampal regions of three consecutive coronal sections visualized using Olympus FV3000 confocal microscope and the images were captured at 10× magnification.
The number and size of MOAB-2 positive Aβ plaques aswell as Iba1-positive areas were quantitated using ImageJ.
30-µm-thick tissue sections were used for microglial
enveloping analysis. The tissue sections were sequentiallypermeabilized with 0.5% Triton X-100 in PBS for 1 h at
RT, blocked in 10% goat serum + 0.5% Triton X-100 in
PBS for 1 h at RT, and probed with indicated primary

antibodies (anti-Iba1, 1:100, Cat. No. sc-32,725, Santacruz biotechnology, anti-lamp1, 1:800, Cat. No. ab24170,
Abcam, diluted in 0.5% Triton X-100 containing 1.5%
goat serum) overnight at 4℃. The sections were thenincubated with Goat Anti-Mouse IgG (1:1000, Cat. No.
115-605-003, Jackson ImmunoResearch Laboratorie),
and Cy3-AffiniPure F (ab′) ₂ fragment Goat anti-Rabbit
IgG (1:1000, Cat. No. 111-166-144, Jackson ImmunoResearch Laboratorie) for 2 h at RT, respectively. Briefly
washed 3X in PBS, the sections were stained with 1%
Thioflavine S for 8 min at RT, followed by washing in 70%
ethanol for 4 min and rinsing with water. The slides were
mounted in Fluoromunt™ Aqueous Mounting Medium(Cat. No. F4680; Sigma-Aldrich) and coverslipped before

Song et al. Journal of Neuroinflammation(2024) 21:167

Page 8 of 19

imaging. The unilateral hippocampal fields of three consecutive coronal sections were visualized using Olympus
FV3000 confocal microscope and the z-stacked images
were captured using a 40 × objective at 1024 × 1024-pixel
resolution at 3 μm z-step. The number and size of ThioSpositive Aβ plaques as well as the number of microglial
cells within 25 μm radius from the center points of theAβ plaques were quantified using ImageJ. The neuritic
dystrophy associated with compact amyloid plaques
was measured using ImageJ according to the method
described by Condello et al., 2015 [30]. A Z-projectionof three optical slices through the center of the plaquewas generated for each of the plaques and was used to
calculate microglia coverage as well as the dystrophic
neuritic area. The colocalization of the plaques and Iba1positive areas was recognized using ImageJ. The intersections between the colocalized areas and the plaqueperimeter, which was defined using ImageJ, were identified manually for each of the plaque. The proportion of
the summed arcs of the perimeter with Iba1 colocalization was defined as microglia coverage. To compare thedystrophic neurite formation in areas with or without
microglia coverage, the angles used to define microglia
coverage were further extended to the limit of Lamp1positive areas (plaque area excluded), which were recognized using ImageJ. The Lamp1-positive areas within thisextended selection as well as the Lamp1-positive areas
outside the selection were calculated, respectively andwere normalized to their respective angular degrees. The
significance of correlation between microglia coverageand Lamp1-positive areas in different size of Aβ plaque
was tested.

Tubulin β3 a neuronal marker and > = 90% of neuronalcells was obtained.
pEGFP-N1 plasmids were then used to transfect neuronal cells alone or co-transfected with indicated oligos
using Lipofectamine2000 to improve visualization ofneuron morphology [33]. Half the medium was replacedwith fresh medium containing either sAPPα (10 nM)
or atRA (2 µM) or vehicles before processed for further analysis. Z-stacked images acquired with Olympus
FV3000 confocal microscope (at 1 μm step, 40 × objective) of at least 30 randomly chosen GFP-positive cells
from each sample were used for Sholl analysis to calculate the number of dendrites intersections on concentric
circles at 5 μm intervals using ImageJ [34].

Mouse embryonic primary cortical cell culture

The amount of Aβ1–42 monomer in cells and tissues
were determined using Amyloid beta 42 Mouse ELISA
Kit (Cat. No. KMB3441, Invitrogen, Carlsbad, CA),according to the manufacturer’s instructions. The hemisphere hippocampal tissues were lysed in RIPA buffer (8µL/ 1 mg of tissue) for 30 min on the ice, followed by centrifugation at 12,000 × g for 30 min. 5 µL supernatant ofeach sample was used for Aβ42 ELISA assay. The cells in
12-well plates were homogenized in RIPA buffer (60 µL
per well) for 30 min on the ice. 50 µL supernatant of eachsample was used for Aβ 42 ELISA assay.

Primary cortical tissues were isolated from E15.5
C57BL/6J embryo [31, 32]. The pregnant female mice(E15.5) were euthanized with CO2 and the embryos werecollected in ice-cold Hank’s balanced salt solution (HBSS,
Invitrogen). The cortical tissues of the embryos were dissected in fresh pre-chilled HBSS under a stereomicroscope, followed by digestion in 0.25% trypsin at 37 °C for
15 min. The cortical tissues were then resuspended inplating medium (BME medium + 10% FBS + 0.45% glucose (20%) + 1% sodium pyruvate + 1% glutamine + 1%
Penicillin/streptomycin) and triturated with care usinga pasteur pipettes until a single-cell suspension was
obtained. Dissociated cells were inoculated in six-wellplates that were coated with poly-L-lysine (1 mg/mL)
and were allowed for settle in an incubator filled with 5%
CO2 at 37 °C for 4 h. The medium was then replaced withneuron medium (Neurobasal medium + 2% B27 + 1% glutamine + 0.5% penicillin/streptomycin). The cell purities
were assessed 24 h later using immunofluorescence with

Cell lines and cultured cell transfections

The human embryonic kidney 293T cells, the mouse hippocampal neuronal cell line HT22 and mouse microglial
cell BV2 were purchased from ATCC (Manassas, Virginia, USA). 293T, HT22 and BV2 cells were cultured in
the Dulbecco’s modified Eagles medium (DMEM) with
10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37℃ in a humidified atmosphere with 5% CO2.
293T and HT22 cells were plated on 6-well plates at thedensity of 4 × 105/ well. The cells were transfected with
50 nM RNAs or 1 µg indicated plasmids using Lipofectamine 2000 transfection reagent according to the
manufacturer’s instructions. The cell culture medium
was replaced with fresh medium containing indicatedtreatments 24 h after transfection and maintained before
processed to further analysis at indicated time points.
ELISA

Golgi staining and dendrite arborization analysis

The FD Rapid Golgi Stain Kit (Cat. No. PK401, FD Neurotechnologies) was used for mouse brain to Golgi staining according to manufacturer’s instruction. Briefly, themouse brain hemispheres (n = 4) obtained immediately
after sacrifice were immersed into mercuric chloride
solution (mixture of solutions A and B) for 2 weeks. The
samples were then transferred to a cryoprotectant solution (solution C) and were kept in dark for another week.

Song et al. Journal of Neuroinflammation(2024) 21:167

The samples were then frozen in isopentane on the dry
ice and stored at -80℃. 100-µm thick serial coronal sections of hippocampus region were obtained using a cryostat microtome (CM1950, Leica). The slices were stainedwith ammonium hydroxide solution (mixture of solution
D and E) for 10 min at room temperature, followed by
2 × brief wash with water. Slices were dehydrated withgraded alcohols and cleared with xylenes, and then coverslipped using neutral balsam (BL704A, Biosharp, Hefei,
China). Z-stack images (at 1 μm steps) of pyramidal neuron from hippocampus CA1 regions were captured under
20 × objective using EVOS FL Auto microscope (Thermo
Fisher Scientific, Waltham, USA) and 3D images werereconstructed using Vaa3D software [35]. 10–15 pyramidal neurons per mouse were selected for Sholl analysis
using ImageJ. The dendrite intersections on concentric
circles with radius increased by 5 μm each starting fromthe cell soma were quantified.
Phagocytosis assay of FITC-conjugated Aβ1–42

FITC-conjugated Aβ1–42 peptide (Cat. No. M212900,
MREDA, Beijing, China) was dissolved in DMSO to
make the 200 µM stock solution and stored at − 80 °C
before use. 1 µM FITC-Aβ42 working solution was made3 days before use by adding DMEM to the stock solution
and incubation at 37 °C to allow for fibrillar Aβ aggregation. HT22 cells were transfected with indicated oligos orconstructs for 12 h, followed by co-culturing with BV2cells for 12 h. Mock-transfected cells were used as controls. Fibrillar Aβ solution was added to the co-cultured
cells to a final concentration of 500 nM and incubatedfor 2 h to allow for Aβ phagocytosis. The cells were thenfixed with 4% PFA at room temperature for 15 min andpermeabilized with 0.2% Triton X-100 in PBS for 10 min
with brief washes before and after. After blocking with
10% goat serum and 2% BSA in PBST for 1 h at room
temperature, the cells were probed with anti-Iba1 (1:500,
Cat. No. 019-19741, Wako, Japan) overnight at 4 °C. Afterwashing with PBST three times, the cells were then incubated with Alexa Fluor 647 conjugate secondary antibody(1:800, Cat. No. 111-606-004, Jackson ImmunoResearch)for 1 h at room temperature. Cell nuclei were stainedwith Hoechst 33342 (1 µg/mL, Cat. No. B2261, SigmaAldrich) at room temperature for 10 min. The z-stackconfocal images were captured at 0.7 μm step using 100×oil immersion objective and the 3D representative images
were created using ImageJ’s 3D Viewer plugin. Nine optical fields were randomly chosen for each experiment andthe z-stack images were captured under 40 × objective at
1 μm step size using Olympus FV3000 confocal microscope (Olympus, FV3000, Tokyo, Japan). The images
were processed with FV31S-SW Viewer software (version 2.5) before quantification analysis of Aβ phagocytosis. The online software cellpose (version 2.0) was used

Page 9 of 19

for cellular segmentation, and ImageJ was used to assess
the number of Aβ+ microglia and analysis the mean fluorescence intensity of FITC-Aβ1–42.
Statistical analysis

All statistical analyses were performed using GraphPad
Prism 8.0 software (GraphPad Software Inc., San Diego,
CA, USA). Comparisons between two groups were performed using a two-tailed unpaired Student’s t-test.
Multiple comparisons were performed using a one-way
ANOVA followed by post hoc test. Data are expressed asmean ± SD or mean ± SEM, as indicated in the relevant
figure legends. A probability of p < 0.05 was considered
statistically significant.

Results
Identification and validation of the differentially expressed
miRNAs associated with AD in blood plasma

The global expression profiles of miRNA in blood plasma
samples taken from 11 AD patients as well as from 14 age
and sex matched cognitively normal volunteers were analyzed using miRNA-seq. After the initial filtering, 1010
known miRNAs were successfully mapped using the reference miRBase (version 20.0) (Table S1). 108 miRNAs
were concurrently expressed in both AD patients andcontrols, with one miRNA, miR-4732-5p, was detected
only in the AD plasma (7 patients). The divergence in gene
expression between AD patients and healthy control wasdemonstrated by principal component analysis (PCA)
with normalized gene expression data (Fig. 1A). Total of
41 (24 upregulated and 17 downregulated) differentiallyexpressed miRNAs (DEMs) with statistical significanceswere identified using two-tailed, unpaired Student’s
t-test (adj. p < 0.05, Fig. 1B, Table S2). The differences inmiRNA profiles of blood plasma from AD patients andcontrols were also demonstrated by the agglomerative
hierarchical clustering of the DEMs using Pearson correlation coefficient and average distance, showing two
main groups identified (p < 0.05, Fig. 1B). Among them,
29 DEMs (16 upregulated and 13 downregulated) has a|log2-fold change| >1 (FDR adjusted p < 0.05) (Fig. 1C). In
addition, the expression of 7 miRNAs (hsa-miR-425-3p,
hsa-miR-192-5p, hsa-miR-1306-5p, hsa-miR-1307-3p,
hsa-miR-150-5p, hsa-miR-423-3p, hsa-miR-4433b-5p) in
the blood plasma samples of all 12 AD patients was out of
the 95% confidence intervals of their levels in the healthy
controls (Table S3). These miRNAs, along with miR4732-5p that was found to be exclusively expressed in the
blood plasma of the AD patients, might be used as potential biomarkers for AD diagnosis and need to be verified. Next, the targets of these potential miRNAs whichwere related to cognitive dysfunction genes were selected(Fig. 1D). The result shown that two upregulated DEMs,
miR-140 and miR-122, which were conserved between

Song et al. Journal of Neuroinflammation(2024) 21:167

human and mouse, came to our notion due to theirpotential functional significance in the dimension of thefundamental AD pathology. They both target ADAM10,
the major α-secretase in CNS that is involved in APP
processing (Fig. 1D). Also, terms of neuronal function,
cell death and homeostasis, etc. were enriched in miRNAtargets, related to cognitive dysfunction genes (Fig. 1E).
The reproducibility of the expression of these two miRNAs was further verified in the blood plasma of an independent test cohort of AD patients and controls, as wellas in the brain parenchyma of 6-month-old APP/PS1
mice and C57BL/6J control mice. The results of quantitative RT-PCR demonstrated the significantly upregulated
expression of miR-140 and miR-122 in the blood plasmaof AD patients, as well as in the hippocampi and cortex
of APP/PS1 mice relative to the controls (Fig. 1E and G).
We proposed that miR-140 and miR-122 might play critical roles in the pathological progression of AD and thepotential mechanisms were further investigated in vitro
and in vivo using APP/PS1 mouse model.
Overexpression of miR-140 and miR-122 reduced nonamyloidogenic processing of APP via targeting ADAM10

miR-140 and miR-122 mimics were transfected either
individually or in combination into mouse hippocampal neuron cells (HT22) as well as into HEK-293T cells.
Knock-down of the endogenous expression of miR-140
and miR-122 in these cells were accomplished by transfection of recombinant pcDNA3.1 plasmids overexpressing the specific inhibitory sequences of miR-140 andmiR-122 “Tough Decoy” (TuD) that contained two copies of miRNA-binding sites of each miRNA (Fig. 2A and
B). Transfection of pcDNA3.1-miR-140-miR-122-TuD
significantly reduced the endogenous expression of miR140 and miR-122 in cultured 293T cells (Fig. 2C). Compared with that of the mock-transfected control cells or
the control cells transfected with scramble RNA, overexpression of miR-140 or miR-122 significantly down-regulated the protein expression of ADAM10 (Fig. 2D and G).
Co-expression of miR-140 and miR-122 mimics further
inhibited the expression of ADAM10 in both 293T and
HT22 cells (Fig. 2D and G). On the contrary, inhibition of
the endogenous miR-140 and miR-122 expression in thesecells by overexpressing TuD containing tandem inhibitors of miR-140 and miR-122 resulted in up-regulated
ADAM10 (Fig. 2D and G). In accordance, accumulated
APP and decreased soluble APPα were demonstrated in
the cells overexpressing miR-140 and miR-122 and viceversa (Fig. 2E, F, H and I). The imbalance between amyloidogenic and non-amyloidogenic processing of APP incells overexpressing miR-140 and miR-122 was likely to
be ascribed to the reduced ADAM10 expression, sinceadministration of retinoic acid, an ADAM10 promoter
inducer, restored the expression of ADAM10 as well as

Page 10 of 19

the α-proteolysis of APP in miR-140/miR-122 overexpressing cells. Although significant changes of expression
were not observed, nonsignificant trend of the expression changes in APP and sAPPα were shown in HT22
cells overexpressing either miR-140 or miR-122 in our
results of western blot assay, indicating a cumulative
effect of miR-140 and miR-122 via targeting the common
downstream target ADAM10 (Fig. 2H and I). Althoughno significant influence on Aβ production was observedin HT22 cells with miR-140/miR-122 overload, depletionof endogenous miR-140 and miR-122 by overexpressing
miR-140-miR-122-TuD significantly reduced Aβ production in HT22, indicating a redundancy in APP processing
and the therapeutic potentials of these miRNAs in AD
treatment (Fig. 2J).
Co-overexpression of miR-140 and miR-122 induced
memory impairments in wild type mice and aggravatedcognitive dysfunction in APP/PS1 mice

Memory impairments along with other AD pathologies
including Aβ deposition, abnormal long-term potentiation appears between 3–6 months in APP/PS1 mice [36].
Recombinant AAV9 virus overexpressing both miR-140
and miR-122 (AAV9-miR140-miR-122-eGFP) were constructed for in vivo functional assays. A total number of
8 × 1010 genome copies AAV9-miR-140-miR-122-eGFP
or control virus AAV9-Scr-eGFP were injected into
both sides of the lateral ventricles in 4-month-old APP/
PS1 mice as well as in 4-month-old wild type C57BL/6J
mice to induce overexpression of miR-140 and miR-122
in widespread regions in mouse brains. Age-matched
untreated mice were used as controls. Expression andregion of infection of the recombinant virus in mousebrains were verified at two months after the injection(Fig. S1). Behavioral test batteries comprised of the Y
maze and Morris water maze were performed two monthafter the injection to assess the short-term working memory and the spatial learning, respectively. Overexpressionof miR-140 and miR-122 induced impaired memory inwild type mice and aggravated cognitive deteriorationin AD mice, demonstrated by the reduced alternationsin the Y maze and reduced number of platform-crossingin the probe trials in water maze, respectively, comparedwith their counterparts injected with AAV9-Scr-eGFP as
well as the controls (Fig. 3A-3D, 3A’-3D’). Administrationof all-trans retinoic acid (20 mg/g), which was shown
to upregulate ADAM10 transcription via binding to thetwo retinoid X-receptor/retinoic acid receptor (RXR/
RAR) in the promoter, significantly increased the alternations in Y maze as well as decreased the escape latencies in the Morris water maze in the AD mice injectedwith AAV9-miR140-miR-122-eGFP, comparing with the
AD mice received vehicle treatment (Fig. 3E, F and F’).
The expression of ADAM10, sAPPα, APP and Aβ in theSong et al. Journal of Neuroinflammation(2024) 21:167

Page 11 of 19

Fig. 3 Overexpression of miR-140 and miR-122 altered proteolytic processing of APP and induced cognitive impairment in mice. (A-B). Behavioral assessment of C57BL/6J mice two months after intracerebroventricular injection of recombinant AAV9 vectors expressing miR-140 and miR-122 or scramble
RNA, showing heatmap of animal’s position in Y maze and trajectory in Morris water maze (A), percentage of alternations in Y maze test (A’), latency to
the first entry of platform (B) and number of crossings (B’) in Morris water maze. The results were presented as means ± SD (n = 13 for C57 mice; n = 10 for
AAV9-Scr-eGFP; n = 10 for AAV9-miR-140-miR-122-eGFP). *p < 0.05, ***p < 0.001. Statistical analyses were performed using two-tailed unpaired two-tailed
unpaired Student’s t-test. (C-D). Behavioral assessment of APP/PS1 mice two months after intracerebroventricular injection of recombinant AAV9 vectors
expressing miR-140 and miR-122 or scramble RNA, showing percentage of alternations in Y maze test (C), latency to the first entry of platform (D) andnumber of crossings (D’) in Morris water maze. The results were presented as means ± SD (n = 10 for each group). *p < 0.05, **p < 0.01. Statistical analyses
were performed using two-tailed unpaired Student’s t-test. (E-F). Behavioral assessment of APP/PS1 mice two months after injected with AAV9-miR-140miR-122-eGFP with or without atRA treatment, showing percentage of alternations in Y maze test (E), latency to the first entry of platform (F) and numberof crossings (F’) in Morris water maze. The results were presented as means ± SD (n = 10 for each group). *p < 0.05. Statistical analyses were performed
using two-tailed unpaired Student’s t-test. (G-I). Results of western blot and densitometric quantification of the relative expression of ADAM10 (G), sAPPα(H) and APP (I) in the hippocampi of control C57 and APP/PS1 mice. C57 and APP/PS1 mice transfected with recombinant AAV9 vector expressing
scramble RNA or miR-140 and miR-122. APP/PS1 mice transfected with AAV9-miR-140-miR-122-eGFP were divided into two groups, which received atRA
treatment or mock treatment, respectively. The expression of β-actin was used as an internal control. The results were presented as means ± SD (n = 3 foreach group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical analyses were performed using One-way ANOVA or two-tailed unpaired Student’s
t-test. (J). ELISA analysis of the level of Aβ42 in the hippocampi of the uninfected APP/PS1 mice as well as APP/PS1 mice transfected with recombinant
AAV9 vector expressing scramble RNA or miR-140 and miR-122. The uninfected C57 mice was used as the controls

hippocampi of these mice were evaluated using western
blot and ELISA assay, respectively. Compared to theircounterparts injected with AAV9-Src-eGFP, the expression of ADAM10 and sAPPα was significantly reducedin the hippocampi of APP/PS1 and C57 mice injectedwith AAV9-miR140-miR-122-eGFP while the levelsof APP were significantly increased only in the hippocampi of APP/PS1 mice overexpressing miR-140 andmiR-122 (Fig. 3G-I). The mice infected with the control

AAV9-Scr-eGFP virus displayed no significant change ofthe levels of these proteins, comparing with those of theuninfected controls. Administration of atRA restored theexpression of ADAM10 and sAPPα while reduced APP
in the brain of APP/PS1 mice injected with AAV9-miR140-miR-122-eGFP (Fig. 3G-I). However, in line withwhat observed in in vitro analysis using cultured cells,
the results of ELISA assay showed no significant change
in the levels of Aβ monomers in the hippocampi of APP/

Song et al. Journal of Neuroinflammation(2024) 21:167

PS1 mice transfected with either AAV-Src-eGFP or AAVmiR-140-miR-122-eGFP (Fig. 3J). These observations
indicated the detrimental effects of increased miR-140
and miR-122 expression in AD pathology via targeting
ADAM10 were unlikely to be ascribed to enhanced complimentary β-cleavage of APP.
Dysregulated miR-140 and miR-122 expression affected
microglial chemotaxis and Aβ phagocytosis

Moreover, the potential influence of dysregulated miR140 and miR-122 expression on APP metabolism andPage 12 of 19

Aβ deposition was further investigated in mice injectedwith AAV9-miR-140-miR-122-eGFP using immunohistochemical analysis with monoclonal anti-Aβ antibodies (MOAB-2) as well as using Thioflavine S staining,respectively. As expected, amyloid plaques were observedin APP/PS1 mice while were negative in wild type mice
(Fig. 4A). Compared with their untransfected counterparts or the controls transfected with AAV9-Src-eGFP,
overexpression of miR-140 and miR-122 did not significantly promote amyloid plaque deposition in either APP/
PS1 mice or wild type mice with stereotactic injection ofFig. 4 Overexpression of miR-140 and miR-122 reduced microgliosis and microglia phagocytosis. (A-B). Representative confocal z-stack images showing
results of immunohistochemical analysis in the DG subregions on coronal sections of the hippocampus of control C57 and APP/PS1 mice, which weretransfected with AAV9-Scr-eGFP or AAV9-miR-140-miR-122-eGFP, respectively. Aβ deposition was stained with MOAB-2 (white) or ThioS (green). Microglia
cells were stained using anti-Iba1 (red). Scale bar: 200 μm. (C-E). Quantitative analysis of the immunofluorescence intensities of three consecutive sectionsof three independent biological replicates, showing average number of MOAB-2+ Aβ plaques (C), ThioS+ fibrilar Aβ plaques (D) and percentage of Iba1
postive area (E). Results were presented as meam ± SEM. *p < 0.05. Statistical analyses were performed using One-way ANOVA. (F-G). Results of western
blot analysis and densitometric quantifications of the relative expression of Iba1 (F) and IL-1β (G) in the hippocampi of APP/PS1 mice transfected with
AAV9-Scr-eGFP or AAV9-miR-140-miR-122-eGFP, respectively. Untransfected APP/PS1 mice and wild type C57 mice were used as controls. The expressionof β-actin was used as an internal control. The results were presented as means ± SD (n = 3). *p < 0.05, **p < 0.01. Statistical analyses were performed using
One-way ANOVA. (H-J). Aβ phagocytosis analysis in BV2 cells co-cultured with untransfected HT22 cells or HT22 cells that were transfected with scramble
miRNAs, mimics of miR-140 and miR-122, pcDNA3.1, or pcDNA3.1-miR-140-miR-122-TuD, respectively. Representative images of immunofluorescenceshowing Iba1 (red) labelled BV2 cells and FITC-Aβ (green) (H). Quantitative analysis of fluorescence intensities showing the average fluorescence intensityof FITC-Aβ in phagocytic BV2 cell (I) and proportion of Aβ phagocytic BV2 cells (J). The results were presented as means ± SEM (n = 9). *p < 0.05, **p < 0.01,***p < 0.001. Statistical analyses were performed using One-way ANOVA. Scale bar: 20 μm. (K-M). Western blot analysis and densitometric quantificationof the relative expression of Cx3cl1 (K), Cx3cr1 (L) and Trem2 (M) in the hippocampi of APP/PS1 mice transfected with AAV9-Scr-eGFP or AAV9-miR-140miR-122-eGFP, respectively. Untransfected APP/PS1 mice and wild type C57 mice were used as controls. The expression of β-actin was used as an internal
control. The results were presented as means ± SD (n = 3). Statistical analyses were performed using One-way ANOVASong et al. Journal of Neuroinflammation(2024) 21:167

AAV9-miR140-miR-122-eGFP, shown by both MOAB-2
and Thioflavine S (ThioS) staining (Fig. 4A-D). In accordance with the increased Aβ deposition in APP/PS1 mice,significantly enhanced microglial activities were demonstrated by the increased immunofluorescence intensities
of Iba1 observed in the hippocampi of APP/PS1 mice aswell as in the hippocampi of APP/PS1 mice transfectedwith AAV9-Scr-eGFP, compared with the control C57
mice (Fig. 4A and E). On the contrary, instead of triggering microgliosis, the results of immunohistofluorescenceshowed significantly reduced Iba1 immunoreactivities in
the hippocampi of APP/PS1 mice overexpressing miR140 and miR-122 (Fig. 4A and E). Results of western
blot analysis confirmed the reduction of Iba1 expression
in the hippocampi of mice overexpressing miR-140 andmiR-122, although the expression of proinflammatory
cytokines IL-1β was dramatically increased (Fig. 4F and
G). These results led to our speculation of defective hippocampal microglia reactivity in the brains of the miceoverexpressing miR-140 and miR-122, where the aggravated neuroinflammation was observed comparing withthat of the control mice. This hypothesis was further
supported by Aβ phagocytosis analysis in vitro. Mimics of miR-140 and miR-122 and pcDNA3.1-miR-140miR122-TuD were overexpressed in cultured HT22 cells,
respectively. Untransfected cells, cells transfected withscramble miRNAs or pcDNA3.1, were used as controls.
These cells were then co-cultured with BV2 cells andthe phagocytotic activities of FITC-labeled Aβ42 were
quantified. Our results showed that overexpressing miR140 and miR-122 in HT22 significantly suppressed thephagocytotic activities in the co-cultured BV2 cells while
downregulation of miR-140/miR-122 expression in HT22
significantly increased the number of phagocytotic cells
as well as phagocytotic activities in the co-cultured BV2cells (Fig. 4H-J). These results were in correlation with
our previous observation of the reduced microglial activities and the increased sizes of Aβ plaques in the hippocampi of mice overexpressing miR-140 and miR-122.
Given that both Fractalkine (CX3CL1) and TREM2,
a highly expressed chemokine on neuronal cells thatmediate neuron-to-glia communications via its receptor (CX3CR1) on microglia and a single-transmembrane
domain protein expressed on microglia that function asa sensor of apolipoproteins in Aβ plaques, were also targeted by ADAM10’s proteolytic cleavage, the expressionof CX3CL1/CX3CR1 and TREM2 was further evaluated in our mouse models [37]. No significant changes inthese proteins’ expression in the hippocampi of APP/PS1
mice overexpressing miR-140/miR-122 were revealed by
the western blot assay, compared with that of the miceoverexpressing scramble RNA and that of the uninfected
control mice (Fig. 4K and M). Collectively, the results
suggested that the restrained microglial chemotaxis/

Page 13 of 19

phagocytosis observed both in vivo and in vitro withupregulated miR-140 and miR-122 expression was independent of dysregulation of these receptors.
The reduced neuroprotective plaque-associated microglia
exacerbated neurite dystrophy around the amyloid plaqueGiven that excessive Aβ cause neuroinflammation andactivate microglia/astroglia to carry out phagocytosis ofAβ plaques [38], the numbers of microglia around Aβ
deposition (immuno-stained with MOAB-2) were calculated within 25 µm diameter from the center of Aβplaques. Significantly reduced numbers of microglia
around Aβ plaques were identified in the dentate gyrus of
AAV9-miR140-miR-122-eGFP infected APP/PS1 mice,compared to their counterparts infected with AAV9Scr-eGFP, indicating the decreased microglial migration
toward Aβ plaques (Fig. 5A and B). The reduced microglial migration in the hippocampi of mice overexpressing
miR-140 and miR-122 was in line with the downregulation of reactive microglia signature, including Chi3l1
and H2-Ea, as shown by the quantitative real-time PCR
analysis (Fig. 5C and C’). 16–40 ThioS labeled hippocampal Aβ fibrils per mouse were subsequently selected. The
microglia coverage over the plaques were subsequently
quantified. Compared with the mice infected with AAV9Scr-eGFP, the overall microglial enveloping of plaques
was significantly reduced in the hippocampi of APP/
PS1 mice overexpressing miR-140/miR-122 (Fig. 5D
and E). Given the neurotoxicity around Aβ aggregates,
we further assessed the neuritic dystrophy within theplaque regions in the mouse brains with or without
miR-140/miR-122 overexpression. Compared with the
APP/PS1 mice infected with AAV9-Scr-eGFP, quantification of the z-stack images of immunolabelled dystrophic neurites using anti-Lamp1 revealed significantly
escalated areas of swollen dystrophic neutites in thejuxta-Aβ plaque regions in mice overexpressing miR-140
and miR-122 (Fig. 5D and F). The neurotoxicity was significantly exacerbated in the sectors outwards microgliauncovered fractions in the APP/PS1 mice overexpressing
miR-140/miR-122 as well as in the control APP/PS1 miceoverexpressing scramble RNA (Fig. 5G and H). Significant negative correlations between the areas of neuritic
dystrophy and the microglia coverage were noticed, especially around the amyloid plaques with diameter smaller
than 9 μm (Fig. 5I). The results indicated the weakened
neuroprotection of microglia barrier within the plaqueadjacent microenvironment in the brains of the micewith dysregulated miR-140 and miR-122 expression. Further assessment of dendrite arborization of well-stained
pyramidal neurons in the ipsilateral hippocampal CA1
regions was performed using Sholl analysis (Fig. 5J).
Compared with that of the mice injected with AAV9Scr-eGFP, quantification of basal dendritic intersections

Song et al. Journal of Neuroinflammation(2024) 21:167

Page 14 of 19

Fig. 5 Overexpression of miR-140 and miR-122 reduced microglia reactivity and increased Aβ plaque neurotoxicity. (A). Representative confocal z-stack
images of immunohistochemical analysis, showing Aβ plaques (MOAB-2, white) and plaque-associated microglia (Iba1, red) in the DG region of untransfected APP/PS1 mice, APP/PS1 mice transfected with AAV9-Scr-eGFP or AAV9-miR-140-miR-122-eGFP, respectively. Nuclei were counterstained with
Hoechst33342 (blue). Scale bar: 20 µm. (B). Quantification of the number of plaque-associated microglia within 25 µm diameter from the center of Aβplaques in the DG subregions on coronal sections of the hippocampi of APP/PS1 mice, APP/PS1 mice transfected with AAV9-Scr-eGFP or AAV9-miR-140miR-122-eGFP, respectively. Results were recorded on three consecutive sections of three independent biological replicates. n > 150 plaques for eachgroup. The median of each group was indicated with the black dash lines. *p < 0.05. Statistical analyses were performed using One-way ANOVA. (C-C’).
Results of quantitative RT-PCR showing the relative expression of Chi3l1 and H2-Ea in the hippocampi of C57BL/6J mice, the APP/PS1 mice, APP/PS1 micetransfected with AAV9-Scr-eGFP and APP/PS1 mice transfected with AAV9-miR-140-miR-122-eGFP, respectively. The results were presented as means ± SD
(n = 3). **p < 0.01. Statistical analyses were performed using One-way ANOVA. (D). Representative confocal z-stack images of immunohistochemical analysis of microglia coverage and neurite dystrophy, showing ThioS+ Aβ plaques (blue), microglia (Iba1, red), and dystrophic neurite (Lamp1, yellow). Scalebar: 20 μm. (E-H). Quantification of the microglia coverage over the plaques (E) and Lamp1 area (F) around each individual plaque. Quantification of thearea of dystrophic neurites extending radially outward from microglia-covered or -uncovered plaque perimeter in the hippocampi of APP/PS1 mice transfected with AAV9-Scr-eGFP (G) or AAV9-miR-140-miR-122-eGFP (H). Results of three consecutive sections of three independent biological replicates were
presented as means ± SEM or means ± SD (n > 70 plaques for each group). ****p < 0.0001. Statistical analyses were performed using two-tailed unpaired
Student’s t-test. (I). Correlation analysis of the total Lamp1 positive area and microglia coverage for each individual plaques, which were grouped into 3
different ranges of sizes plaques. Over 70 plaques were selected from APP/PS1 mice transfected with AAV9-Scr-eGFP (n = 3) or AAV9-miR-140-miR-122eGFP (n = 3), respectively. (J). Golgi-Cox staining showing pyramidal neurons in the hippocampal CA1 of APP/PS1 mice transfected with AAV9-Scr-eGFPor AAV9-miR-140-miR-122-eGFP, respectively. Scale bar: 20 μm. (K-L). Sholl analysis of the dendric intersections of pyramidal neurons. The number of basal
dendritic intersections (K) and the area under the curve (L) were quantified. Results are expressed as means ± SD. ****p < 0.0001. Statistical analyses were
performed using two-tailed unpaired Student’s t-test

on concentric circles drawn from the cell body at 5 μm
intervals revealed reduced dendritic extension in the hippocampi of mice overexpressing miR-140 and miR-122,
indicating the exacerbated neuronal hypotrophy (Fig. 5K
and L).

The neurotoxicity of miR-140 and miR-122 overexpression
was alleviated by additional sAPPαGiven that overexpression of miR-140/miR-122 significantly reduced α-proteolysis of APP both in vitro and invivo without significant influence on Aβ deposition, we
further asked whether the neuritic dystrophy and dysregulated microglial chemotaxis induced by excessive
miR-140/miR-122 were due to mitigated sAPPα protection. The primary cortical neuron cells were preparedSong et al. Journal of Neuroinflammation(2024) 21:167

from E15.5 mouse embryos. The primary neurons weretransfected with miR-140 and miR-122 mimics, followed by treatment with recombinant sAPPα, anti-Aβ42
blockade or atRA, respectively. The control cells weretransfected with scramble RNA, followed by mock treatments. GFP-positive cells were randomly selected for
dendrite arborization analysis. Neurons transfectedwith mimics of miR-140 and miR-122 showed diminution of dendritic length and reduced in dendritic complexity compared with that of the neurons transfected

Page 15 of 19

with scramble RNA (Fig. 6A-6A’’). However, comparedwith the cells transfected with the expression vectors,
increased dendritic length and complexity were observedin neuronal cells with downregulated endogenous miR140 and miR-122 by overexpressing miR-140-miR-122TuD (Fig. 6A and A’’). The reduced number of dendritic
branches and the outgrowth of total neurite in neurons
transfected with mimics of miR-140 and miR-122 were
restored by addition of sAPPα and atRA, respectively,
while depletion of Aβ with anti-Aβ blockade showed

Fig. 6 Neurite outgrowth and microglia phagocytosis impaired by miR-140 and miR-122 overexpression was restored by administration of sAPPα in vitro.
(A-D). Representative fluorescence images of primary cortical neuron cells transfected with mimics of miR-140 and miR-122, pcDNA3.1, or pcDNA3.1-miR140-miR-122-TuD (A), respectively, followed by treatment of atRA (B), sAPPα (C) and Aβ blockage (D). pEGFP was co-transfected for visualization. Cells
transfected with GFP constructs and followed by mock treatment were used as controls. (A’-D’). Quantification of the dendric intersections and area under
the curve using Sholl analysis. The results were presented as means ± SEM (n > 30 neurons for each group). *p < 0.05, **p < 0.01, ****p < 0.0001. Statisticalanalyses were performed using One-way ANOVA. (E-E’’). Analysis of Aβ phagocytosis in BV2 cells co-cultured with HT22 cells that were transfected withscramble miRNAs with or without administration of 10 nM sAPPα. Representative immunofluorescence images (E) of microglia Aβ phagocytosis, showing BV2 cells (anti-Iba1, red) and FITC-Aβ (green). Quantification of Aβ phagocytosis, showing internalized fluorescence intensity of FITC-Aβ per BV2 cell(E’) and the proportion of Aβ phagocytic BV2 cells (E’’). (F-F’’). Analysis of Aβ phagocytosis in BV2 cells co-cultured with HT22 cells that were transfectedwith mimics of miR-140 and miR-122 with or without administration of 10 nM sAPPα. Representative immunofluorescence images (F) of microglia Aβphagocytosis, showing BV2 cells (anti-Iba1, red) and FITC-Aβ (green). Quantification of Aβ phagocytosis, showing internalized fluorescence intensity of
FITC-Aβ per BV2 cell (F’) and the proportion of Aβ phagocytic BV2 cells (F’’). (G). 3D representations of confocal z-stack images of microglia Aβ phagocytosis. The results were presented as means ± SEM (n = 9). Scale bar: 20 μm. *p < 0.05, ***p < 0.001. Statistical analyses were performed using two-tailed
unpaired Student’s t-test

Song et al. Journal of Neuroinflammation(2024) 21:167

minimal improvements in dendrite arborization in thesecells (Fig. 6B and B’’, 6 C–6 C’’, 6D-6D’’). These results
indicated that the adverse effects of overexpression of
miR-140 and miR-122 were likely to be ascribed to the
reduced production of sAPPα due to downregulation of
ADAM10.
Given the reduced microglia migration and coverage over Aβ plaque observed in the hippocampi of miceoverexpressing miR-140/miR-122 as well as the reducedAβ phagocytosis in BV2 cells co-cultured with neuronal cells overexpressing miR-140/miR-122, the potential
beneficiary effects of sAPPα on microglia phagocytosis
was evaluated in vitro. We found that additional sAPPαsignificantly increased the phagocytic activities of FITClabeled Aβ42 in BV2 cells, compared with that of the
mock-treated control cells (Fig. 6E-6E’’). In addition,
administration of sAPPα also restored the impaired
phagocytotic capacities the microglial cells co-cultured
with HT22 cells transfected with miR-140 and miR-122
mimics, although the number of phagocytotic cells was
not affected (Fig. 6F and F’’, 6G). The results collectively
suggested that the reduced production of neuroprotective sAPPα was a key player in the elevated Aβ neurotoxicity and exacerbated the progression of AD-like
pathology in mice with dysregulated miR-140 and miR122 expression.

Discussion
Human brain expresses more than 70% of microRNAs, aberrant expression of which have been shown toassociate with the pathological progression of AD bymodulating amyloidogenesis and Tau pathology, as wellas unknown molecular pathways [39]. The differently
expressed microRNAs may be reflected in the profiles ofcirculating small non-coding RNAs and serve as prognostic biomarkers for AD diagnosis at early stages [40].
For instance, miR-206, targeting brain-derived neurotrophic factor (BDNF) and miR-29, targeting β-site amyloid precursor protein cleaving enzyme 1 (BACE1), wereamong the most addressed microRNA signatures ofAD [41, 42]. In this study, the levels of 16 miRNAs were
found significantly increased in the blood plasma of AD
patients. Among them, the plasma levels of 7 miRNAs,
were outside of the 95% confidence intervals of theirhealthy controls, along with miR-4732-5p that exclusively
detected in AD plasma, formed an 8-member group ofmiRNA signature that might be used for AD biomarker
candidates. Although not consistently changed in allplasma samples of AD patients, the two significantly
upregulated miRNAs, miR-140 and miR-122, both targeting ADAM10, the major form of α-secretase in brain,came to our notion due to the mechanistic dimensions.
Overexpression of miR-140 and miR-122 in CNS induced
cognitive impairment in wild-type mice and exacerbated

Page 16 of 19

cognitive dysfunction in APP/PS1 mice. Although neither significant changes in the number and size of amyloid plaques nor significantly increased levels of overallAβ42 were observed in these mice, compared to theiruntransfected counterparts and counterparts transfectedwith control virus, significantly decreased sAPPα production associated with miR-140/miR-122 overexpression and thus downregulated ADAM10 were verified
both in vitro and in vivo. These results indicated that
although the ADAM10 mediated α-proteolysis of APP
was likely to be the primary source of sAPPα production, alternative pathways, including ubiquitylation andother α-/β-like cleavage may play redundant roles in APP
metabolism [43]. In addition, given that the majority of
APP is retained in Golgi and only 10% of APP eventuallytransported to the plasma membrane, it was not surprising that downregulation of ADAM10 by overexpressing
miR-140 and miR-122 had minor influence in the level of
total cellular APP [44].
There is ongoing controversy regarding the correlation between Aβ levels and cognitive function in thepathogenesis of AD [45]. Although Aβ buildup in brain
was one of the earliest molecular pathological changes
detected even decades before AD symptoms, the progression of Aβ deposition showed weak correlation, if
any, with the severities of this condition [46]. The twomost relevant forms of β-amyloid peptides are Aβ40
and Aβ42, which are more likely to form self-aggregates.
Among numerous Aβ species, including Aβ monomer,
oligomer, protofibrils and fibrils, Aβ oligomer and protofibrils are considered toxic while Aβ monomers havebeen shown to be neuroprotective [47]. Oligomeric Aβ,Aβ protofibrils and Aβ deposits activate microglia andinduce inflammatory response via inflammasome signaling as well as chemokine receptors [48]. Excessive Aβ andtau also interfere with regulated autophagy, induce proapoptotic proteins, and eventually lead to neuronal death,
which in turn, aggravate neuroinflammation by releasing
damage-associated molecular patterns (DAMPs). On theother hand, microglia express various chemokine receptors that can be activated by Aβ to initiate chemotaxis,
a cellular event that is important for microglia mediated Aβ clearance by phagocytosis and hydrolysis [49].
Nonetheless, uncontrolled microglia reaction can result
in destructive neuroinflammation, which is one of theneuropathological hallmarks of AD [50]. Besides passive
response induced by Aβ and damaged neurites, microglia
cells were suggested to play causative roles in early AD
pathogenesis. Many genes within the risk loci for AD, e.g.
TREM2 and CD33, have been found expressed actively
in microglia and affect immunosurveillance, which areessential for maintaining CNS homeostasis [49]. On thecontrary to the activated microgliosis around Aβ plaques
that commonly seen in AD-related neuroinflammation,

Song et al. Journal of Neuroinflammation(2024) 21:167

the number of microglia cells in DG as well as within
the 25 μm radius plaque-juxtaposed area were decreased
in mice overexpressing miR-140 and miR-122. Further
analysis revealed significant reduction of the microglia
coverage over compact ThioS+ Aβ plaques in the miceoverexpressing miR-140/miR-122, which was correlated with the significantly increased neurotoxicity andneurite dystrophy. Given that neuron-derived CX3CL1
is also cleaved by ADAM10 to release soluble form of
CX3CL1, which serves as chemoattractant, we proposedthat CX3CL1 was less cleaved in mice overexpressing
miR-140/miR-122 due to downregulated ADAM10 withresultant decrease in chemotactic CX3CL1 and increasein the membrane-bound suppressive CX3CL1. However, neither decreased soluble CX3CL1 nor increased
static form of CX3CL1 was observed in the mice overexpressing miR-140 and miR-122 in the brain. Therefore,
the proteolytic cleavage of this chemokine in the brainsof the mice overexpressing miR-140 and miR-122 waslikely to be maintained by functional redundancies of
disintegrins and metalloproteinase including ADAM10
and ADAM17 [37]. Likewise, western blot analysis of the
microglial specific immunoreceptor TREM2 (triggering
receptor expressed on myeloid cell 2), which could also
be cleaved by ADAM10, demonstrated no significant
change regarding it’s cleavage in the brain tissues of themice overexpressing miR-140 and miR-122. Given that
ADAM10 principally acts at the cell surface, downregulation of ADAM10 in neuronal cells may have limited
impact on the cleavage of microglial TREM2 [51].
sAPPα, the non-amyloidogenic product of ADAM10,
has been well characterized for its neuroprotective roles
and its therapeutic potentials in Alzheimer’s disease [52].
It has been shown to protect neuronal survival in adverse
conditions both in vitro and in vivo [16, 53]. ExogenoussAPPα or sAPPα overexpression have been shown to prevent AD pathology and improve learning and memories
in AD mouse models [54–56]. sAPPα also displayed neurotrophic activities in promoting the proliferation andneurite outgrowth of neural progenitor cells (NPC), possibly via activating ERK and MAP-kinase signaling pathways [15, 57]. In this study, we found that transfection of
mimics of miR-140 and miR-122 significantly reduced
the mouse embryonic neural cells neurite outgrowth,which could be rescued by additional sAPPα but not byanti-Aβ antibody neutralization. Likewise, the attenuatedmicroglial chemotaxis-phagocytosis in cultured BV2 cells
transfected with mimics of miR-140 and miR-122, including both the percentage of phagocytic microglia and thephagocytic index, was also restored by exogenous sAPPα.
Previous research demonstrated that sAPPα interactedwith microglial receptors, such as class A SR, LRP1 andGroup II mGluR, to enhance Aβ phagocytosis [58, 59].
As a dominant strategy in AD intervention, the rationale

Page 17 of 19

underlying a causative role for Aβ in AD pathogenesis
has been constantly challenged, despite of the recent
approval of lecanemab in AD therapy [60].

Conclusions
Two miRNAs, miR-140 and miR-122, were found significantly increased in the peripheral blood samples of AD
patients as well as in the brain tissues of APP/PS1 mice.
Overexpression of miR-140 and miR-122 in the hippocampus induced cognitive impairment in wild type C57
mice and exacerbated cognitive deterioration in APP/
PS1 mice. Given that ADAM10, the major α-secretasein CNS, is the common downstream target of miR-140
and miR-122, the potential influence of dysregulated
miR-140/miR-122 expression on APP metabolism andAD-related pathologies have been studied in mice withectopic hippocampal miR-140 and miR-122 expression
as well as in vitro. Our results suggested that reduced
α-proteolytic cleavage of APP and the consequential
reduced production of sAPPα was likely to be the key
player in mediating the pathological outcomes of dysregulated expression of miR-140 and miR-122 in CNS, byshowing unaltered soluble Aβ levels and Aβ deposits in
APP/PS1 mice overexpressing miR-140/miR-122 and thatadministration of RA to induced ADAM10 expression
significantly improved the performance of these mice inbehavioral assessment. In addition, either overexpressionof miR-140/miR-122-specific TuD or treatment of RA
or sAPPα, but not Aβ depletion, could improve the suppressed neurite outgrowth of embryonic neuronal cells
transfected with mimics of miR-140 and miR-122 as wellas the reduced Aβ phagocytosis in co-cultured microglia.
Our results support a promising strategy in AD therapy
by manipulating multiple targets in the α-proteolytic
pathway of APP.
Abbreviations
AD	Alzheimer’s diseaseAβ	Accumulated beta-amyloid
APP	Amyloid precursor proteinssAPPα	Soluble APPαatRA	
All-trans retinoic acid
MMSE	Mini-Mental State Examination
MWM	Morris water maze
TMM	Trimmed mean of M-values
TPM	Transcripts per million
GO	Gene Ontology
KEGG	Kyoto Encyclopedia of Genes and Genomes
PCA	Principal component analysis
DEMs	Differentially expressed miRNAs
BDNF	Brain-derived neurotrophic factor
BACE1	β-site amyloid precursor protein cleaving enzyme 1
DAMPs	Damage-associated molecular patterns
TREM2	Triggering receptor expressed on myeloid cell 2
NPC	Neural progenitor cells

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03162-z.

Song et al. Journal of Neuroinflammation(2024) 21:167

Supplementary Material 1
Supplementary Material 2

Page 18 of 19

2.
3.

Supplementary Material 3
Supplementary Material 4
Supplementary Material 5
Acknowledgements
Not applicable.
Author contributions
CS conducted bioinformatic analysis, western blot assays, in vitro analysis
of embryonic neuronal neurite outgrowth and microglia phagocytosis. SFL
constructed the mouse models, performed behavioral assessment, TuD
design, target gene expression analysis, Golgi staining and immunohistology
assays. CS and SFL prepared figures. YRM conducted the clinical study. LPL and
GKD performed the image analysis. YZ and XSL performed behavioral tests
and western blot assay. OMZ and YW assist mouse work and data processing.
LBZ supervised the experimental design and helped data analysis. JL directed
the clinical study. YZ supervised the project, conceived this research, drafted
the paper, designed the experiment, and interpreted the data. All authors
reviewed the manuscript.

4.
5.

6.
7.
8.
9.
10.

Funding
This work was sponsored by Guangdong Provincial Key R&D Program(#2023B1111050011) and the Natural Science Foundation of Guangdong(#2023A1515010293).

11.

Data availability
No datasets were generated or analysed during the current study.

12.
13.

Declarations
Ethics approval and consent to participate
The clinical trial study was approved by the ClinicalTrials.gov (ID:
NCT03653156). All patients gave written informed consent. All mice werehoused at temperature (21–25℃) under 12 h- light/dark cycle with free accessto water and food. All animal experiments were approved by the Institutional
Animal Care and Use Committee of Jinan University (IACUC-20230625-02).
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.

14.
15.

16.
17.

Author details
1
School of Life Science and Technology, Jinan University, Guangzhou,
Guangdong 510632, China
2
Department of Neurology, Institute of Neuroscience, Key Laboratoryof Neurogenetics and Channelopathies of Guangdong Province andthe Ministry of Education of China, The Second Affiliated Hospital,
Guangzhou Medical University, Guangzhou, Guangdong 510260, China
3
Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministryof Education CNS Regeneration Collaborative Joint Laboratory, Jinan
University, Guangzhou, Guangdong 510632, China

18.

Received: 24 April 2024 / Accepted: 26 June 2024

21.

19.
20.

22.
References
1. Montine TJ, Koroshetz WJ, Babcock D, Dickson DW, Galpern WR, Glymour MM,
Greenberg SM, Hutton ML, Knopman DS, Kuzmichev AN, et al. Recommendations of the Alzheimer’s disease-related dementias conference. Neurology.
2014;83:851–60.

23.
24.

Rajmohan R, Reddy PH. Amyloid-Beta and Phosphorylated Tau Accumulations Cause Abnormalities at synapses of Alzheimer’s disease neurons. J
Alzheimers Dis. 2017;57:975–99.
Copenhaver PF, Kögel D. Role of APP interactions with Heterotrimeric G
Proteins: physiological functions and pathological consequences. Front Mol
Neurosci. 2017;10:3.
Chow VW, Mattson MP, Wong PC, Gleichmann M. An overview of APP processing enzymes and products. Neuromolecular Med. 2010;12:1–12.
Roher AE, Lowenson JD, Clarke S, Woods AS, Cotter RJ, Gowing E, Ball MJ.
beta-Amyloid-(1–42) is a major component of cerebrovascular amyloid
deposits: implications for the pathology of Alzheimer disease. Proc Natl Acad
Sci U S A. 1993;90:10836–40.
Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress
and problems on the road to therapeutics. Science. 2002;297:353–6.
Asamu MO, Oladipo OO, Abayomi OA, Adebayo AA. Alzheimer’s disease: therole of T lymphocytes in neuroinflammation and neurodegeneration. Brain
Res. 2023;1821:148589.
Cheng Y, Tian DY, Wang YJ. Peripheral clearance of brain-derived Aβ in
Alzheimer’s disease: pathophysiology and therapeutic perspectives. Transl
Neurodegener. 2020;9:16.
Ries M, Sastre M. Mechanisms of Aβ clearance and degradation by glial cells.
Front Aging Neurosci. 2016;8:160.
Heo C, Chang KA, Choi HS, Kim HS, Kim S, Liew H, Kim Ja, Yu E, Ma J, Suh YH.
Effects of the monomeric, oligomeric, and fibrillar Aβ42 peptides on the
proliferation and differentiation of adult neural stem cells from subventricularzone. J Neurochem. 2007;102:493–500.
Stein TD, Anders NJ, DeCarli C, Chan SL, Mattson MP, Johnson JA. Neutralization of transthyretin reverses the neuroprotective effects of secreted amyloid
precursor protein (APP) in APPSW mice resulting in tau phosphorylation andloss of hippocampal neurons: support for the amyloid hypothesis. J Neurosci.
2004;24:7707–17.
Fahrenholz F. Alpha-secretase as a therapeutic target. Curr Alzheimer Res.
2007;4:412–7.
Suh J, Choi Se H, Romano Donna M, Gannon Moira A, Lesinski Andrea N, Kim
Doo Y, Tanzi Rudolph E. ADAM10 missense mutations Potentiate β-Amyloid
Accumulation by impairing Prodomain Chaperone function. Neuron.
2013;80:385–401.
Tanzi RE, Moir RD, Wagner SL. Clearance of Alzheimer’s Abeta peptide: themany roads to perdition. Neuron. 2004;43:605–8.
Gakhar-Koppole N, Hundeshagen P, Mandl C, Weyer SW, Allinquant B, Müller
U, Ciccolini F. Activity requires soluble amyloid precursor protein alpha topromote neurite outgrowth in neural stem cell-derived neurons via activation of the MAPK pathway. Eur J Neurosci. 2008;28:871–82.
Dar NJ, Glazner GW. Deciphering the neuroprotective and neurogenic
potential of soluble amyloid precursor protein alpha (sAPPα). Cell Mol Life Sci.
2020;77:2315–30.
Lammich S, Kojro E, Postina R, Gilbert S, Pfeiffer R, Jasionowski M, Haass C,
Fahrenholz F. Constitutive and regulated α-secretase cleavage of Alzheimer’samyloid precursor protein by a disintegrin metalloprotease. Proc Natl Acad
Sci. 1999;96:3922–7.
Kuhn PH, Wang H, Dislich B, Colombo A, Zeitschel U, Ellwart JW, Kremmer E,
Roßner S, Lichtenthaler SF. ADAM10 is the physiologically relevant, constitutive α-secretase of the amyloid precursor protein in primary neurons. EMBO J.
2010;29:3020–32.
Peron R, Vatanabe IP, Manzine PR, Camins A, Cominetti MR. Alpha-Secretase
ADAM10 Regulation: Insights into Alzheimer’s Disease Treatment. Pharmaceuticals (Basel) 2018, 11.
Lee HR, Shin HK, Park SY, Kim HY, Lee WS, Rhim BY, Hong KW, Kim CD.
Cilostazol suppresses β-amyloid production by activating a disintegrin andmetalloproteinase 10 via the upregulation of SIRT1-coupled retinoic acidreceptor-β. J Neurosci Res. 2014;92:1581–90.
Endres K, Fahrenholz F, Lotz J, Hiemke C, Teipel S, Lieb K, Tüscher O, Fellgiebel
A. Increased CSF APPs-α levels in patients with Alzheimer disease treatedwith acitretin. Neurology. 2014;83:1930–5.
Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficientalignment of short DNA sequences to the human genome. Genome Biol.
2009;10:1–10.
Kozomara A, Griffiths-Jones S. miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res. 2014;42:D68–73.
Robinson MD, Oshlack A. A scaling normalization method for differential
expression analysis of RNA-seq data. Genome Biol. 2010;11:1–9.

Song et al. Journal of Neuroinflammation(2024) 21:167

25. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008;5:621–8.
26. Stocks MB, Moxon S, Mapleson D, Woolfenden HC, Mohorianu I, Folkes L,
Schwach F, Dalmay T, Moulton V. The UEA sRNA workbench: a suite of tools
for analysing and visualizing next generation sequencing microRNA andsmall RNA datasets. Bioinformatics. 2012;28:2059–61.
27. Chen Y, McCarthy D, Robinson M, Smyth GK. edgeR: differential expression
analysis of digital gene expression data user’s guide. Bioconductor User’s
Guide; 2014.
28. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package fordifferential expression analysis of digital gene expression data. bioinformatics
2010, 26:139–140.
29. Haraguchi T, Ozaki Y, Iba H. Vectors expressing efficient RNA decoys achieve
the long-term suppression of specific microRNA activity in mammalian cells.
Nucleic Acids Res. 2009;37:e43.
30. Condello C, Yuan P, Schain A, Grutzendler J. Microglia constitute a barrier thatprevents neurotoxic protofibrillar Aβ42 hotspots around plaques. Nat Commun. 2015;6:6176.
31. Sciarretta C, Minichiello L. The preparation of primary cortical neuron cultures
and a practical application using immunofluorescent cytochemistry. Methods Mol Biol. 2010;633:221–31.
32. Beaudoin GM 3rd, Lee SH, Singh D, Yuan Y, Ng YG, Reichardt LF, Arikkath J.
Culturing pyramidal neurons from the early postnatal mouse hippocampus
and cortex. Nat Protoc. 2012;7:1741–54.
33. Roussignol G, Ango F, Romorini S, Tu JC, Sala C, Worley PF, Bockaert J, Fagni L.
Shank expression is sufficient to induce functional dendritic spine synapses
in aspiny neurons. J Neurosci. 2005;25:3560–70.
34. George J, Baden DG, Gerwick WH, Murray TF. Bidirectional influence of
sodium channel activation on NMDA receptor-dependent cerebrocorticalneuron structural plasticity. Proc Natl Acad Sci U S A. 2012;109:19840–5.
35. Peng H, Ruan Z, Long F, Simpson JH, Myers EW. V3D enables real-time 3D
visualization and quantitative analysis of large-scale biological image data
sets. Nat Biotechnol. 2010;28:348–53.
36. Trinchese F, Liu S, Battaglia F, Walter S, Mathews PM, Arancio O. Progressive
age-related development of Alzheimer-like pathology in APP/PS1 mice. Ann
Neurol. 2004;55:801–14.
37. Pawelec P, Ziemka-Nalecz M, Sypecka J, Zalewska T. The impact of the
CX3CL1/CX3CR1 Axis in Neurological disorders. Cells 2020, 9.
38. Bolmont T, Haiss F, Eicke D, Radde R, Mathis CA, Klunk WE, Kohsaka S, Jucker
M, Calhoun ME. Dynamics of the microglial/amyloid interaction indicate arole in plaque maintenance. J Neurosci. 2008;28:4283–92.
39. Bandakinda M, Mishra A. Insights into role of microRNA in Alzheimer’s
disease: from contemporary research to bedside perspective. Int J Biol Macromol. 2023;253:126561.
40. Kanach C, Blusztajn JK, Fischer A, Delalle I. MicroRNAs as Candidate Biomarkers for Alzheimer’s Disease. Noncoding RNA 2021, 7.
41. Lee ST, Chu K, Jung KH, Kim JH, Huh JY, Yoon H, Park DK, Lim JY, Kim JM, Jeon
D, et al. miR-206 regulates brain-derived neurotrophic factor in Alzheimer
disease model. Ann Neurol. 2012;72:269–77.
42. Hébert SS, Horré K, Nicolaï L, Papadopoulou AS, Mandemakers W, Silahtaroglu AN, Kauppinen S, Delacourte A, De Strooper B. Loss of microRNA cluster
miR-29a/b-1 in sporadic Alzheimer’s disease correlates with increased BACE1/
beta-secretase expression. Proc Natl Acad Sci U S A. 2008;105:6415–20.
43. Wang H, Saunders AJ. The role of ubiquitin-proteasome in the metabolism ofamyloid precursor protein (APP): implications for novel therapeutic strategies
for Alzheimer’s disease. Discov Med. 2014;18:41–50.
44. Vetrivel KS, Thinakaran G. Membrane rafts in Alzheimer’s disease beta-amyloid production. Biochim Biophys Acta. 2010;1801:860–7.
45. Duyckaerts C, Potier M-C, Delatour B. Alzheimer disease models andhuman neuropathology: similarities and differences. Acta Neuropathol.
2008;115:5–38.

Page 19 of 19

46. Murphy MP, LeVine H. 3rd: Alzheimer’s disease and the amyloid-beta peptide.
J Alzheimers Dis. 2010;19:311–23.
47. Hampel H, Hardy J, Blennow K, Chen C, Perry G, Kim SH, Villemagne VL, Aisen
P, Vendruscolo M, Iwatsubo T, et al. The Amyloid-β pathway in Alzheimer’s
Disease. Mol Psychiatry. 2021;26:5481–503.
48. Horibe S, Emoto T, Mizoguchi T, Tanaka T, Kawauchi S, Sasaki N, Yamashita
T, Ikeda K, Emoto N, Hirata KI, Rikitake Y. Endothelial senescence alleviates cognitive impairment in a mouse model of Alzheimer’s disease. Glia.
2024;72:51–68.
49. Althafar ZM. Targeting Microglia in Alzheimer’s Disease: From Molecular
Mechanisms to Potential Therapeutic Targets for Small Molecules. Molecules
2022, 27.
50. Choi SH, Tanzi RE. Adult neurogenesis in Alzheimer’s disease. Hippocampus.
2023;33:307–21.
51. Lau SF, Fu AKY, Ip NY. Receptor-ligand interaction controls microglial chemotaxis and amelioration of Alzheimer’s disease pathology. J Neurochem.
2023;166:891–903.
52. Khezri MR, Mohebalizadeh M, Ghasemnejad-Berenji M. Therapeutic potentialof ADAM10 modulation in Alzheimer’s disease: a review of the current
evidence. Cell Commun Signal. 2023;21:60.
53. Corrigan F, Vink R, Blumbergs PC, Masters CL, Cappai R, van den Heuvel C.
sAPPα rescues deficits in amyloid precursor protein knockout mice following
focal traumatic brain injury. J Neurochem. 2012;122:208–20.
54. Obregon D, Hou H, Deng J, Giunta B, Tian J, Darlington D, Shahaduzzaman
M, Zhu Y, Mori T, Mattson MP, Tan J. Soluble amyloid precursor protein-α
modulates β-secretase activity and amyloid-β generation. Nat Commun.
2012;3:777.
55. Deng J, Habib A, Obregon DF, Barger SW, Giunta B, Wang YJ, Hou H, Sawmiller
D, Tan J. Soluble amyloid precursor protein alpha inhibits tau phosphorylation through modulation of GSK3β signaling pathway. J Neurochem.
2015;135:630–7.
56. Tan VTY, Mockett BG, Ohline SM, Parfitt KD, Wicky HE, Peppercorn K,
Schoderboeck L, Yahaya MFB, Tate WP, Hughes SM, Abraham WC. Lentivirusmediated expression of human secreted amyloid precursor protein-alphaprevents development of memory and plasticity deficits in a mouse modelof Alzheimer’s disease. Mol Brain. 2018;11:7.
57. Rohe M, Carlo AS, Breyhan H, Sporbert A, Militz D, Schmidt V, Wozny C,
Harmeier A, Erdmann B, Bales KR, et al. Sortilin-related receptor with
A-type repeats (SORLA) affects the amyloid precursor protein-dependent
stimulation of ERK signaling and adult neurogenesis. J Biol Chem.
2008;283:14826–34.
58. Habib A, Sawmiller D, Tan J. Restoring soluble amyloid precursor protein αfunctions as a potential treatment for Alzheimer’s Disease. J Neurosci Res.
2017;95:973–91.
59. Durand D, Turati J, Rudi MJ, Ramírez D, Saba J, Caruso C, Carniglia L, vonBernhardi R, Lasaga M. Unraveling the β-amyloid clearance by astrocytes:
involvement of metabotropic glutamate receptor 3, sAPPα, and class-A
scavenger receptor. Neurochem Int. 2019;131:104547.
60. Ayton S, Bush AI. β-amyloid: the known unknowns. Ageing Res Rev.
2021;65:101212.

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