{"paper_id":"458aa98e-9481-4d74-b8cd-e4461eaa901f","body_text":"Effects of ADAM10 deletion on APP shedding, synapse and synaptic plasticity in adult mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report Effects of ADAM10 deletion on APP shedding, synapse and synaptic plasticity in adult mice Qiulan Wei, Shuihui Liu, Yan Liang, Jianlong Zhuang, Peng Li, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8193475/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A disintegrin and metalloproteinase 10 (ADAM10) is a member of the ADAMs (a deintegrin and metalloproteinases) family, which hydrolyzes various cellular receptors and signaling molecules (such as APP) to regulate the development of body organs and tissues. As ADAM10 is a susceptibility gene for Alzheimer's disease (AD), we generated adult neural cell-specific ADAM10 conditional knockout (ADAM10 cKO) mice to study its effects on APP shedding, synaptic integrity, and cognitive function. Our results revealed that ADAM10 deletion significantly increased levels of sAPPβ, C99, total Aβ peptides, while decreasing levels of sAPPα and C83 in the hippocampus and cortex. These changes trigger neuroinflammation, as evidenced by elevated GFAP expression. Furthermore, synaptophysin expression was reduced in the hippocampus and cortex, with postsynaptic density protein-95 (PSD-95) showing decreased expression in the hippocampal CA1 region. Synaptic ultrastructure exhibited abnormalities, and long-term potentiation (LTP) induction was impaired, ultimately leading to learning and memory deficits. These findings advance our understanding of ADAM10's biological functions and provide mechanistic insights for AD prevention and therapeutic strategies. deintegrin and metalloproteinase 10 (ADAM10) amyloid precursor protein (APP) synapses long-term potentiation (LTP) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1 INTRODUCTION Alzheimer's Disease (AD), which mainly occurs in individuals aged 65 or older, is one of the most common neurodegenerative diseases. This disease is one of the sixth leading causes of death worldwide, and its incidence continues to rise annually, creating a heavy burden for society and families [ 1 ]. The amyloid precursor protein (APP), a type I transmembrane protein associated with AD, primarily mediates physiological and pathological processes through proteolytic cleavage fragments, including the amyloid-beta (Aβ) peptide [ 2 ]. Amyloid plaques, one of the hallmark, pathological features in the brain tissue of AD patients, and are predominantly composed of Aβ peptides [ 3 ][ 4 ]. Aβ peptide is generated by the APP through the amyloidogenic cleavage pathway. In the amyloidogenic cleavage pathway, the ectodomain shedding of the APP ,which is firstly done by β -secretase, generates the soluble APPβ (sAPPβ) and the C-terminal fragment 99 (C99). Subsequently, the cleavage of C99 generates Aβ peptides of varying lengths (38–49 amino acids) and APP intracellular domains (AICD) by γ-secretase. However, another proteolytic processing pathway exists for APP. In this pathway, APP shedding is firstly done by α-secretase but not by β-secretase, and the site of hydrolysis is within Aβ peptide (at the lysine16-leucine17 bond), generating the soluble APPα (sAPPα) and the C-terminal fragment 83 (C83). Then, the cleavage of C83 releases small P3 fragment and AICD by γ-secretase. Thereby APP cleavage by α-secretase can prevent Aβ formation [ 2 ][ 4 ][ 5 ]. It has now been confirmed that amyloid plaques do not contain P3, and sAPPα has neurotrophic and neuroprotective effects [ 6 ][ 7 ][ 8 ]. Disntegrin/metalloproteinase 10 ( ADAM10 ), a member of the ADAMs (a deintegrin and metalloproteinases) family, is widely expressed in mammalian cells and mediates the hydrolysis of various cellular receptors and signaling molecules (e.g., APP and Notch) [ 13 ]. ADAM10 has been identified as the principal constitutive α-secretase responsible for APP processing in neuronal cells. Notably, downregulation of ADAM10 gene expression in primary cultured neurons significantly reduces sAPPα production, while enhancing sAPPβ and Aβ igeneration [ 9 ]. However, a study by Jorissen E et al. demonstrated that neuronal ADAM10 deletion in neural precursor cell conditional knockout mice not only decreases C83 and sAPPα, but also paradoxically reduces sAPPβ, C99, total Aβ, Aβ40, and Aβ42 [ 10 ]. Intriguingly, additional evidence suggests that α-secretase and β-secretase activities are not strictly interdependent in primary cortical neurons. Specifically, inhibition of β-secretase activity could improve the activity of α-secretase, but inhibition of α-secretase activity does not improve the activity of APP cleavage by β-secretase [ 11 ]. All data mentioned above come from in vitro studies. However, a recent in vivo study showed that ADAM10 gene expression is significantly downregulated in the cerebral cortex of ADAM10 cKO mice, leading to reduced sAPPα and elevated sAPPβ and endogenous Aβ production [ 12 ]. These findings suggest that α-secretase competes with β-secretase for APP cleavage in vivo. Consequently, enhancing α-secretase activity could not only suppress Aβ generation but also promote sAPPα production, potentially offering a dual therapeutic strategy for treating or even preventing AD. Synapses are specialized intercellular junctions between neurons or between neurons and non-nerve cells (e.g.,muscle cells, gland cells). They serve as critical sites for both neuronal connectivity and information transmission. Generally the presynaptic terminal typically exhibits spherical swelling containing numerous synaptic vesicles and mitochondrion. The thickened axonal membrane forms the presynaptic membrane, which harbors voltage-gated, calcium ion channels, and membrane fusion proteins (e.g., SNAP25, Syntaxin). Synaptic cleft is located between the pre- and postsynaptic membranes, it contains various adhesion molecules essential for signal transduction. Synapses are classified into type I and type Ⅱ based on differences in the electron-dense band thickness of the pre- and postsynaptic membranes. The type I synapses (excitatory synapses) exhibit asymmetric pre- and postsynaptic membranes, with the postsynaptic membrane being thicker than the presynaptic membrane. The type Ⅱ synapses (inhibitory synapses) characterized by symmetrical pre- and postsynaptic membranes of comparable thickness. Synaptic plasticity is that synapses dynamically adapt their functional efficiency (e.g., long-term potentiation [LTP] or depression [LTD]) and morphology (e.g., postsynaptic density [PSD] thickening, synaptic cleft narrowing) in response to activity. For example, tetanic stimulation of hippocampal synapses induces LTP-a sustained increase in excitatory postsynaptic potential (EPSP) amplitude།which is pivotal for studying cognitive memory mechanisms [ 14 ]. Many neurodegenerative diseases show pathological features of synapses, including abnormal expression of synaptic proteins, synaptic loss, synaptic dysfunction, and structural alterations of synapses [ 15 ][ 16 ]. Therefore, we used the ADAM10 conditional knockout (cKO) mouse model established by our research group to investigate the effects of ADAM10 gene on APP metabolism [ 17 ], aiming to elucidate the interplay the relationship between ADAM10 α-secretase and β-secretase in the substrate APP cleavage in vivo. Additionally, we examined the synaptic-related protein, synaptic ultrastructure and hippocampus LTP in ADAM10 cKO mice to further explore the effects of ADAM10 gene on synaptic function in adult mice. These findings advance our understanding of the ADAM10’s physiological and pathological functions. 2 MATERIALS AND METHODS 2.1 MATERIALS Antibodies and Kits Antibodies used for Western blot were shown in Table 2. HRP AffiniPure goat anti-rabbit IgG (H + L) ( E030120 ) was purchased from EarthOx, LLC·San Francisco, CA·USA. HRP AffiniPure goat anti-mouse IgG (H + L) (EM35110) was purchased from Beijing Emarbio Science & Technology Co.,LTD.. Mouse Aβ40 ELISA Kit (E-EL-M0067c) and mouse Aβ42 ELISA kit (E-EL-M0068c) were purchased from Elabscience. The mouse monoclonal GFAP antibody (CBL412C3) was purchased from Millipore. An anti-mouse Cy3-conjugated secondary antibody kit was purchased from Beyotime. High-sensitivity ECL chemiluminescence kit was purchased from Heron Bio Inc. SABC immunohistochemical staining kit (SA1021) was purchased from Wuhan Doctor De Bio Co., LTD.. Mouse high-sensitivity two-step immunohistochemical detection kit (PV9002) was purchased from Zhongshan Jinqiao Biological Co., LTD.. DAB kit was purchased from Beijing Zhuang Meng International Biological Gene Technology Co.,LTD.. BCA kit (P0012S) was purchased from Beyotime. PCR primers were synthesized by Shanghai Sangon Biological Engineering Technology Service Co., LTD.. Table 2 A list of antibodies were used for Western blot. Target Antibody(Catalog No.) company Total Aβ Anti-β-Amyloid antibody(B-4,sc-28365) Santa Cruz Biotechnology Total FL-APP,total sAPP, mAPP, imAPP, sAPPα and sAPPβ Anti-Alzheimer Precursor Protein A4, a.a 66–81 of APP antibody(22C11) Millipore C99,C83 Anti-Amyloid Precursor Protein C-terminal antibody(A8718) Sigma Aβ37, Aβ38, Aβ39, Aβ40 and Aβ42/43 β-Amyloid, 1–16 Monoclonal Antibody(6E10, SIG-39320) Covance β-actin Anti-β-actin Mouse Monoclonal antibody(E021020) EarthOx, LLC·San Francisco, CA·USA synaptophysin Anti-synaptophysin Antibody(SC-17750) Santa Cruz Biotechnology PSD-95 Anti-PSD-95 Antibody(MA1-045) Thermo Fisher β-actin Anti-β-actin (MJ3007) earth Experimental Animals ADAM10 loxP/loxP transgenic mice were provided by Professors Yuan Zhuang and Xiaohui Wu, Institute of Developmental Biology and Molecular Medicine, Fudan University. CAMKIIα-Cre transgenic mice(on a C57BL/6 pure background) were prepared and identified by our research group. Clean grade C57BL/6J mice were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences. All experimental mice were raised by the Laboratory Animal Center, Fujian Medical University. In accordance with ARRIVE guidelines, the method we used for euthanasia of mice was cervical dislocation. The experimental procedures for the use and care of animals were approved by the Ethics Committee of Fujian Medical University, China. All experiments were performed in accordance with relevant guidelines and regulations. 2.2 METHODS 2.2.1 Generation and genotype analysis of ADAM10 cKO mice CaMKⅡα-Cre/ADAM10 -/ + mice were generated by crossing CaMKⅡα-Cre transgenic mice with ADAM10 loxP/loxP transgenic mice. Subsequently, ADAM10 cKO mice were obtained by breeding CaMKIIα-Cre/ADAM10 -/ +mice with ADAM10 loxP/loxP transgenic mice. Tail DNA Extraction: Genomic DNA was isolated from mice 10–14 days postnatally. PCR Amplification: DNA samples were amplified using primers F1/R1 and KOF/EKOB (see Table 1). Electrophoresis: Mouse genotypes were confirmed by agarose gel electrophoresis. Control groups: ADAM10 loxP/loxP mice , or wild-type C57BL/6J mice. 2.2.2 Western blotting ADAM10 cKO mice, ADAM10 loxP/loxP mice and wild-type C57BL/6J mice were selected for Western blot analysis. The mice were euthanized via cervical dislocation, and then whole brains were rapidly dissected. The cortex, hippocampus and cerebellum were isolated from the brain. Then total protein was extracted using a lysis buffer. Protein concentrations were quantified using a BCA kit (P0010s, Biyuntian Biotechnology Co., LTD.). Protein (20–60µg per lane) were separated on 8% SDS-PAGE gels, 10% SDS-PAGE gels, 16% Tricine-SDS-PAGE gels, and 16% Tricine-SDS-PAGE + 6M Urea gels, and transferred to polyvinylidene difluoride (PVDF, Bio-Rad) membranes for western blot analysis. Band intensities were quantified using ImageJ software (National Institutes of Health). .Statistical comparisons between groups were performed using Student’s t-test. 2.2.3 ELISA(enzyme-linked immunosorbent assay) Proteins were extracted from the forebrain cortex and hippocampus of twelve-month-old ADAM10 cKO mice and ADAM10 loxP/loxP control mice, followed by Western blot analysis. Total protein concentration was determined using a BCA kit (P0010s, Biyuntian Biotechnology Co., LTD.). We used ELISA to detect the concentrations of Aβ40 and Aβ42 in the extracted proteins. The Mouse Aβ40/Aβ42 ELISA Kits (Elabscience) employ a double antibody sandwich method. Experiments were performed according to the manufacturer’s instructions. Optical density (OD) values were measured at 450nm using a microplate reader, Aβ40/Aβ42 concentrations were directly proportional to the OD 450 values, and calculated by plotting standard curves. 2.2.4 Immunohistochemistry Twelve-month-old ADAM10 cKO mice, ADAM10 loxP/loxP control mice and wild-type C57BL/6J control mice were were used for these experiments. The mice were euthanized via cervical dislocation, the brains were rapidly dissected and fixed in 4% paraformaldehyde for 24 hours. Brain tissues were Paraffin-embedded, and 5 µm-thick coronal sections were prepared. We performed immunohistochemical staining. For Aβ staining, we used a SABC kit, a mouse anti-β-amyloid antibody(B-4) and a goat anti-mouse secondary antibody. Sections were developed with 3,3'-diaminobenzidine (DAB) and counterstained with hematoxylin. For synaptophysin staining, we used a SABC kit, an anti-synaptophysin antibody (SC-17750) and a goat anti-mouse secondary antibody. For PSD-95 staining, we used a high-sensitivity two-step kit and an anti-PSD-95 antibody (MA1-045). After dehydration and xylene-based clearing, sections were sealed with neutral resin and dried at 37℃. Images were acquired using a bright-field microscope, and mean optical density was quantified with Image-Pro Plus 6.0.We compared the differences in protein expression between the two groups. 2.2.5 Synaptic ultrastructure We used transmission electron microscopy(TEM) to compare ultrastructure differences between experimental and control mice. Twelve-month-old ADAM10 cKO mice and wild-type C57BL/6J control mice were used in this study. The mice were euthanized via cervical dislocation and their skulls were opened to expose the brain. 4% paraformaldehyde solution was immediately applied to the brain tissue, which was rapidly dissected isolate the hippocampal CA1 regio. Tissue blocks from the hippocampal CA1 region were trimmed to approximately 1 × 1 × 1 mm³ for TEM processing. The tissue blocks were pre-fixed with 3% glutaraldehyde and 1.5% paraformaldehyde in 0.1 M PBS (pH7.2) for ≥ 2 hours at 4℃, followed by three washes with 0.1 M PBS (pH7.2). Post-fixation was performed using 1% osmium tetroxide and 1.5% potassium ferrocyanide in PBS for 1.5 hours at 4°C, after which the tissues were rinsed three times with PBS. The samples were then dehydrated through a graded ethanol series, infiltrated, and embedded in Epoxy Resin 618. Ultrathin sections (100 nm thickness) were prepared, double-stained with uranyl acetate and lead citrate, and imaged using a Philips 208 TEM. Excitatory synapses were identified based on established ultrastructural criteria[18]: a thickened postsynaptic density (PSD), presence of densely stained bands in the postsynaptic membrane, and spherical synaptic vesicles. For quantitative analysis, PSD thickness and synaptic cleft width were measured using stereological methods with square test grids (side length: 86.67 nm) Synaptic interface curvature was calculated as the ratio of the arc length (A) to the chord length (B) of the synaptic membrane. All measurements were analyzed using Image-Pro Plus 6.0 software. 2.2.6 Hippocampal LTP experiment The nine-month-old ADAM10 cKO mice and wild-type C57BL/6J control mice were used for electrophysiological experiments. Hippocampal brain slices (400 µm thick) were prepared according to experimental protocols. The slices were incubated for 2–3 hours prior to recording. For recordings, a single incubated slice was transferred to a semi-immersion thermostatic bath (32°C) on a nylon mesh. A 95% O₂/5% CO₂ gas mixture was continuously perfused above the slice, while non-oxygenated artificial cerebrospinal fluid (ACSF) was irrigated beneath it at a constant rate (1–2 ml/min). Under an operating microscope, a bipolar tungsten stimulation electrode (bare tip) was positioned on the Schaffer collateral pathway in the hippocampal CA3 region. The electrode was connected to a stimulator via an isolator, with stimulation parameters set to 100–300 µs pulse width, 0.1–0.6 mA intensity, and 2–10 s interstimulus intervals. A recording electrode was placed in the CA1 pyramidal cell layer to measure evoked potential. Signals were amplified using a microelectrode amplifier, then routed to a memory oscilloscope, a computer-based data acquisition system, and analytical software.This setup allowed real-time visualization of evoked potentials on both the oscilloscope and computer for simultaneous recording and analysis. 2.2.7 Immunofluorescence detection of glial fibrillary acidic protein (GFAP) expression Twelve-month-old ADAM10 cKO mice and wild-type C57BL/6J control mice were were used for these experiments. The mice were euthanized via cervical dislocation, the brains were rapidly dissected and fixed in 4% paraformaldehyde for 24 hours. 5 µm-thick frozen sections were prepared. For immunofluorescence staining, we incubated the brain sections with a mouse monoclonal GFAP antibody (Millipore) followed by a fluorophore-conjugated secondary antibody. Nuclei were counterstained with DAPI. The slides were mounted using anti-fade mounting mediumto prevent fluorescence quenching. All procedures were performed under minimal light exposure to avoid photobleaching. Confocal microscopy was used for imaging and analysis. By comparing the fluorescence intensity between the experimental (cKO) and control groups, we could directly assess GFAP expression levels. 2.2.8 Data processing Statistical significance was calculated by Student’s t-test using SPSS17.0 software. Data were expressed as (± s), *P < 0.05, **P < 0.01, ***P < 0.001. 3 Results 3.1 Generation genotype analysis of ADAM10 cKO mice Genotype analysis of progeny mice by gel electrophoresis revealed distinct amplification patterns (see Fig. 1). In Cre-transgenic mice, the amplified Cre gene fragment measured 975 bp, whereas wild-type controls showed no detectable bands (see Fig. 1A). Wild-type ADAM10 alleles produced 1,200-bp bands, while ADAM10 alleles with LoxP markers generated 1,000-bp bands (see Fig. 1B).. Mice harboring both ADAM10 loxP/loxP and Cre genes were selected for subsequent experiments. 3.2 Effects on APP processing of deletion of the ADAM10 gene Twelve-month-old ADAM10 cKO mice and ADAM10 loxP/lox P control mice were analyzed. The epitope recognition sites of APP-specific antibodies and their binding schematics are illustrated in Fig. 2O. For the protein amount, the target protein-to-β-actin ratios were included in statistical analyses to control for intra-group variations in sample loading. The results showed that compared with the controls, sAPPα levels decreased by 8.38% (P < 0.001, n = 4 per group) and 10.15% (P < 0.01) in the cortex and hippocampus, respectively(see Fig. 2G-H); C83 levels declined by 13.98% (P < 0.01, n = 4 per group) and 18.93% (P < 0.01) in the cortex and hippocampus, respectively (see Fig. 2I-J); sAPPβ levels increased by 10.31% (P < 0.05, n = 4 per group) and 12.54% (P < 0.001) in the cortex and hippocampus, respectively (see Fig. 2G-H); C99 levels increased by 16.64% (P < 0.01, n = 4 per group) and 21.55% (P < 0.001) in the cortex and hippocampus, respectively(see Fig. 2I-J); and total Aβ levels increased by 30.98% (P < 0.01, n = 4 per group) and 58.97% (P < 0.001) in the cortex and hippocampus, respectively(see Fig. 2K-L). However, total full-length APP (total FL-APP), total soluble APP (total sAPP), mature APP (mAPP), and immature APP (imAPP) were not different between the two groups in the cerebral cortex and hippocampus (see Fig. 2A-F). We used 16% Tricine-SDS-PAGE + 6 M Urea gels to isolate Aβ peptides of different lengths. The results showed that compared with the controls, Aβ40 levels in the cortex and hippocampus of ADAM10 cKO mice increased by 9.60% (P < 0.05, n = 4 per group) and 17.91% (P < 0.01), respectively; Aβ42/43 increased by 31.70% (P < 0.01, n = 4 per group) and 76.93% (P < 0.001) in the cortex and hippocampus of ADAM10 cKO mice, respectively. There was no significant difference in Aβ37, Aβ38, or Aβ39 between the two groups (see Fig. 2M-N). We utilized ELISA to quantify Aβ40 and Aβ42 levels in the cerebral cortex and hippocampus of ADAM10 cKO mice and ADAM10 loxP/loxP control mice. The experimental results showed that compared with the controls, Aβ40 levels in the cortex and hippocampus of ADAM10 cKO mice increased by 17.91% (P < 0.05, n = 4 per group) and 39.53% (P < 0.01), respectively. Similarly, Aβ42 in the cerebral cortex and hippocampus increased by 31.70% (P < 0.01, n = 4 per group) and 76.93% (P < 0.001), respectively (see Fig. 3). These ELISA findings were consistent with the Western blotting results. Immunohistochemistry was employed to assess amyloid plaque deposition in ADAM10 cKO mouse brains. Brain sections from ADAM10 loxP/loxP control mice and ADAM10 cKO mice at 7-,9-,11-, 13-, and 15-month-old (three pairs per group) were stained with mouse anti-β-amyloid monoclonal antibody (B-4) to detect Aβ peptides in the cerebral cortex and hippocampus.The results demonstrated that neither transgenic mouse strain exhibited amyloid plaque deposition. However, soluble Aβ peptides were observed within neuronal cells of both groups.Notably, compared to controls, ADAM10 cKO mice displayed more intense staining of Aβ products in cortical and hippocampal neurons, with a pronounced age-dependent progression in staining intensity (see Fig. 4, Fig. 5)。 3.3 The expressions of synaptophysin and PSD-95 were analyzed by Western blot and immunohistochemistry Western blot was performed to detect synaptophysin and PSD-95 levels in the cortex and hippocampus. For the protein amount, the target protein-to-β-actin ratios were included in statistical analyses to control for intra-group variations in sample loading. The results showed that, compared with the controls, synaptophysin levels in cortex and hippocampus of ADAM10 cKO mice significantly reduced by 24.75% (P < 0.05, n = 3 per group) and 40.10% (P < 0.01), respectively(see Fig. 6A-B). In contrast, PSD-95 levels showed a 23.37% decrease in hippocampus of ADAM10 cKO mice (P < 0.05, n = 3 per group), with no significant differences observed incortical or cerebellar PSD-95 levels between the two groups (see Fig. 6C-D). Immunohistochemical results showed that the synaptophysin was a brown-yellow granular, punctate, or linear immunoreactive deposits with lamellar distributions in hippocampal CA1, CA3, gyrus (DG), and cerebral cortex. Compared with the controls, the experimental groups exhibited synaptophysin immunoreactivity with reduced density, pale staining, and diminishied structures (see Fig. 7). MOD analysis demonstrated significant decreases in synaptophysin levels in ADAM10 cKO mice: 18.78% reduction in CA1 (P < 0.001, n = 3 per group), 14.00% in CA3 (P < 0.01), 21.17% in DG (P < 0.01), and 24.59% reduction in cerebral cortex (P < 0.05) (see Fig. 9A). PSD-95 immunoreactivity presented as brown-yellow granular deposits in the hippocampus CA1, CA3 and the cerebral cortex (see Fig. 8). In experimental groups, CA1 showed lighter-stained PSD-95 granules compared to controls. MOD analysis indicated 26.23% decrease in hippocampal CA1 PSD-95 content (P < 0.05, n = 3 per group), whereas cortical and CA3 regions showed no significant differences (P > 0.05, n = 3 per group) (see Fig. 9B). These findings were consistent with previous Western blot results. 3.4 Synaptic ultrastructure and hippocampal LTP The ultrastructure of synapses is shown in Fig. 10A-B. In the synaptic ultrastructure, mitochondria and numerous spherical synaptic vesicles can be observed in the presynaptic region. Clearly distinguishable presynaptic and postsynaptic membranes, along with the synaptic cleft, are visible. In some synapses, the postsynaptic membrane is noticeably thicker than the presynaptic membrane. Quantitative analysis revealed that excitatory synapses in ADAM10 cKO mice displayed significantly thinner PSD (P < 0.05), reduced synaptic interface curvature (P < 0.05), and widened synaptic clefts (P < 0.05) compared to wild-type C57BL/6J controls (n = 3 per group, see Table 3). These findings indicate that ADAM10 deletion induces synaptic ultrastructural abnormalities and impairs synaptic plasticity in hippocampal neurons of ADAM10 cKO mice. The hippocampal LTP experiment serves as a key physiological correlate of synaptic plasticity, providing mechanistic insights into behavioral regulation. The experimental results shown that baseline LTP was successfully induced in both groups prior to high-frequency stimulation (HFS). Following HFS, ADAM10 cKO mice exhibited significantly reduced field potential slopes and amplitudes. These findings revealed that LTP induction was significantly inhibited after 30min(see Fig. 10C-E). 3.5 The expression of GFAP gene in the cerebral cortex and hippocampus of ADAM10 cKO mice We selected 12-month-old ADAM10 cKO mice and wild-type C57BL/6 control mice, using GFAP-specific labeling to mark radial glial cells and astrocytes in the mouse brain. The results are shown in Fig. 11. Compared with the control group, the GFAP fluorescence intensity was enhanced in the cerebral cortex and hippocampus of ADAM10 cKO mice. These experimental results indicate that after the deletion of the ADAM10 gene, GFAP expression increased in the cerebral cortex and hippocampus of the mice, accompanied by astrocyte proliferation. 4 DISCUSSION We used our ADAM10 cKO mouse model to investigate the function of ADAM10 in adult mice. ADAM10 gene deletion in the forebrain cortex and hippocampus, with higher knockout efficiency in the hippocampus compared to the cortex, demonstrating spatial specificity [ 17 ]. Johannes Prox et al. previously generated a brain-specific ADAM10 cKO model using the CaMKⅡα-Cre promoter[ 12 ], which we also employed. However,phenotypic differences (e.g., survival rate, life span, epilepsy incidence) exist between the two models, likely due to variations in Cre gene copy number, genomic insertion site, and other undefined factors. Notably, Our ADAM10 cKO mice exhibit significantly lower epilepsy incidence and mortality, along with extended lifespans (some surviving > 2 years), compared to the Prox et al. model. In our model, epilepsy does not manifest post-weaning and typically emerges after 10 months of age. Initial seizures are brief and sporadic, but frequency and duration increase progressively with age. These phenotypic advantages make our ADAM10 cKO model particularly suitable for studying Alzheimer’s disease pathogenesis, as chronic neurodegeneration studies require prolonged survival. So, we used our ADAM10 cKO mouse model to investigate ADAM10’s role in APP shedding. Aβ peptides are constitutively secreted by nearly all neuronal cells and non-neuronal cells expressing APP. Among these, Aβ40, Aβ42, and Aβ43 represent the primary neurotoxic species, with Aβ42 and Aβ43 exhibiting enhanced fibrillation propensity. Aβ peptides are the primary component of senile plaques. Current evidence suggests that excessive production of Aβ peptides or impaired clearance pathways leading to amyloid-beta deposition is a critical mechanism underlying AD pathogenesis [ 19 ]. Our data demonstrate decreased sAPPα and C83 levels, along with increased the levels of sAPPβ, C99, total Aβ, Aβ40 and Aβ42/43 in the cortex and hippocampus of the ADAM10 cKO mice. The immunohistochemistry results showed an increase in soluble Aβ levels within neurons in the cerebral cortex and hippocampus of ADAM10 cKO mice. This may be attributed to reduced α-secretase activity of ADAM10, coupled with upregulated β-secretase activity, which shifts APP processing toward β-cleavage. However, our experimental data cannot yet rule out disrupted APP processing or clearance, or altered APP subcellular localization. These findings indirectly suggest that there may be a competitive relationship between ADAM10 α-secretase and β-secretase for APP substrate processing in mice. Synapses play a role in the transmission and transformation of information in neural circuits. To investigate the impact of ADAM10 deletion on synaptic function, we analyzed synaptophysin and PSD-95 expression in the cerebral cortex and hippocampus of the ADAM10 cKO mice, assessed synaptic ultrastructure in the hippocampus, and measured LTP in hippocampal neurons. Synaptophysin,a 38 kDa transmembrane glycoprotein with four transmembrane domains, serves as a presynaptic vesicle marker and is involved in the formation and exocytosis of synaptic vesicles [ 20 ]. It regulates neurotransmitter release, a process vital for interneuronal communication. Hubert Rehm et al. demonstrated that synaptophysin acts as the primary calcium-binding protein of synaptic vesicles and plays an important role in the release of neurotransmitters [ 21 ]. Additionally, synaptophysin interacts with synaptobrevin to form a complex that facilitates functional SNARE assembly, thereby regulating synaptic efficacy [ 22 ]. As a presynaptic terminal marker, synaptophysin is widely used in studies of neurodegenerative diseases (e.g., Parkinson’s disease, AD, and dementia) and transgenic models. Its investigation provides insights into synaptic dysfunction and potential therapeutic strategies. Notably, hippocampal synaptophysin levels are significantly reduced in AD patients, correlating with severe cognitive deficits [ 23 ]. Similarly, Oakley et al. reported age-dependent synaptophysin decline and memory impairment in transgenic mice overexpressing familial AD-linked APP/PS1 mutations [ 24 ]. The study by Cao Y et al. demonstrated that synaptophysin levels are reduced in both AD patients and transgenic mice carrying the amyloid precursor mutation (AβPP) 670/671 (AβPPSWE). Furthermore, bilateral hippocampal injection of Aβ42 in mice resulted in decreased synaptophysin expression at both protein and mRNA levels, accompanied by learning and spatial memory deficits [ 25 ]. Our findings in the ADAM10 cKO mouse model suggest that reduced synaptophysin expression in the cortex and hippocampus of these mice impairs interneuronal communication, thereby contributing to their learning and memory dysfunction. The primary role of postsynaptic element is to receive neurotransmitters released from presynaptic terminals. A uniform, dense layer of material called the postsynaptic density (PSD) is observed on the cytoplasmic surface of the postsynaptic membrane. And PSD-95 is a key scaffolding protein located in the PSD. PSD-95 consists of of three PDZ domains, Src homology 3 (SH3) domain, and a non-catalytic guanylate kinase domain [ 26 ]. Through it’s PDZ domains, PSD-95 binds to glutamate receptors, anchoring glutamate them to the PSD organizing downstream signaling molecules around these receptors [ 27 ]. Thus, PSD-95 is critical for synaptic signal transduction and functional integrity. At present, studies on PSD-95 expression in AD reveal conflicting results. Gylys et al.,reported decreased PSD-95 levels in the cerebral cortex of AD patients [ 28 ] while Love et al. similarly observed reduced PSD-95 in the cortical regions [ 29 ]. In contrast, Leuba et al. found elevated PSD-95 expression in the entorhinal cortex of AD patients [ 30 ], and Gong et al. reported increased PSD-95 in synaptosomes from AD brains [ 31 ]. In transgenic animal models, PSD-95 expressionvaries regionally. SAMP10 mice (a brain aging model) exhibit reduced cortical PSD-95 but unchanged levels in the brainstem [ 32 ]. Similarly, in 5XFAD and JNPL3 AD mouse models, PSD-95 decreases in the apical dendrites of hippocampal CA1 neurons in 6-month-old 5XFAD and 14-month-old JNPL3 mice, yet increases in neuronal soma [ 33 ]. These dynamics suggest PSD-95 expression changes dynamically during disease progression. Reduced PSD-95 may reflect synaptic vulnerability to Aβ peptide toxicity [ 34 ], while compensatory upregulation could indicate de novo synthesis to mitigate synaptic damage [ 35 ]. In our study on the ADAM10 cKO mouse model, we found no significant difference in the expression level of PSD-95 in the cortex and hippocampal CA3 region, while the expression of PSD-95 in the hippocampal CA1 region was decreased. Additionally, levels of Aβ40 and Aβ42 increased in the hippocampus in the hippocampus, along with abnormal synaptic structures. In the hippocampus of ADAM10 cKO mice, the accumulation of Aβ increased. Aβ can strongly bind to the neuronal receptor PirB, triggering intercellular signaling processes that erode neuronal synapses, leading to synaptic contact impairment or even loss. Ultimately, this results in reduced PSD-95 levels in the hippocampal CA1 and impaired learning and memory abilities in the mouse model. Synaptic plasticity refers to property that that allows for more lasting changes in synaptic efficacy, encompassing structural plasticity and functional plasticity, which are interrelated and mutually influence. The plasticity of synaptic structure is manifested as changes in synaptic morphology, including the curvature of the synaptic interface, the thickness of the synaptic dense zone, the width of the synaptic cleft, the synaptic number, and the density of synaptic area. The plasticity of synaptic function is manifested by the enhancement or weakening of synaptic information transmission capacity, such as LTP. Studies of synaptic ultrastructure in the hippocampal CA1 region of a mouse model of neurofibromatosis type I (a neurological dysfunction disorder) have shown that synaptic interface curvature is lower than in normal mice, underscoring the importance of synaptic interface curvature in synaptic plasticity, learning and memory function [ 36 ]. Cerebral vascular hypoperfusion experiments in mice revealed the spatial memory impairment and altered synaptic ultrastructure in the hippocampal CA1 region after 30 days [ 37 ]. Studies of the presenilin (PS) conditional knockout mouse model demonstrated impaired synaptic plasticity, learning and memory, along with inhibited LTP induction in multiple brain regions [ 38 ]. Therefore, our study investigating synaptic ultrastructure and LTP induction in the hippocampal CA1 region of the ADAM10 cKO mice may provide further insights into the mechanisms underling learning behavior in these mice.Our fingdings demonstrate ultrastructural synaptic abnormalities and impaired LTP in hippocampal neurons of ADAM10 cKO mice. The hippocampal CA1 region as the terminal hub of excitatory synaptic circuits, plays a critical role in memory-related synaptic networks. Dysfunction in this region contributes to memory impairment and neurological disorders, which may explain why the hippocampus CA1 region is particularly vulnerable to pathological factors associated with dementia. Regarding the mechanistic basis of these observations, ADAM10 may interact with synaptic-associated protein 97 (SAP97) to regulate synaptic function and formation. Genetic deletion of ADAM10 resulted in synaptic dysfunction, manifested by reduced expression levels of synaptophysin and PSD-95, as well as ultrastructural synaptic abnormalities. These defects likely represent key contributors to the spatial learning and memory deficits observed in the ADAM10 cKO mice. Astrocytic proliferation is a pathological hallmark of structural lesions in the central nervous system (CNS) [ 39 ]. When the CNS suffers severe damage or disease, an inflammatory response may occur, accompanied by the process of astrocytic proliferation. This process is primarily characterized by upregulated GFAP protein expression, hypertrophy of glial cells, and an increase in their numbers [ 40 ]. Astrocytes can accumulate large amounts of Aβ peptides derived from neuronal cells and degrade them in response to localized neurodegeneration [ 41 ][ 42 ]. Studies on transgenic mice overexpressing Aβ peptides in neurons have demonstrated the neurotoxicity of Aβ peptides, showing extensive neuronal degeneration alongside increased glial cell proliferation, suggesting that neuronal loss is caused by apoptosis [ 43 ]. Our study revealed that significant upregulation of GFAP expression was observed in both the cerebral cortex and hippocampus, indicating astrocyte activation. Despite the absence of amyloid plaque deposition, neurons exhibited increased accumulation of soluble Aβ species. Notably elevated levels of neurotoxic Aβ40 and Aβ42 were detected in these regions, with evident oligomerization. This Aβ accumulation triggered neuroinflammatory responses and reactive astrogliosis, ultimately leading to neuronal damage and degeneration. Hippocampal CA1 neurons showed reduced PSD thickness. LTP magnitude was significantly attenuated following high-frequency stimulation. Morris water maze tests revealed impaired spatial learning and memory in our ADAM10 cKO mice, and quantitative stereology demonstrated reduced granule cell numbers in the hippocampal formation [ 44 ]. These findings suggest the Aβ-mediated neuroinflammatory cascade in ADAM10 cKO mice manifests through: (i) astrocyte activation (↑GFAP), (ii) synaptic ultrastructural defects (↓PSD thickness), (iii) functional plasticity impairment (↓LTP magnitude), and (iv) ultimately, cognitive decline. Certainly, in future studies, we will need to further clarify the direct relationship between Aβ, neuroinflammation, and synaptic damage through experiments examining Aβ synaptic localization and inflammation modulation-mediated restoration of synaptic marker expression. In summary, we provided evidence in vivo that ADAM10 gene deletion has remarkable effects on APP processing in the adult mouse brain. The ADAM10 gene is essential for the formation and maintenance of synaptic plasticity. It’s deletion results in synaptic abnormalities,which severely affect information transmission between neurons, leading to impaired learning and memory in adult mice. These results will help us deepen our understanding of ADAM10’s function and provide a theoretical basis for the prevention and treatment of AD. Abbreviations ADAM10 A disintegrin and metalloprotease10 APP Amyloid precursor protein cKO conditional knock out DG dentate gyrus min minute ELISA enzyme linked immunosorbent assay s APPβ soluble APPβ s APPα soluble APPα C99 C-terminal fragment 99 C83 C-terminal fragment 83 AICD APP intracellular domains Aβ Amyloid-beta LTP long-term potentiation PSD postsynaptic dense AD Alzheimer's Disease EPSP excitatory postsynaptic potential TEM transmission electron microscopy MOD mean optical density HFS total FL-APP total sAPP mAPP imAPP high-frequency stimulus total full-length APP total soluble APP mature APP immature APP GFAP g lial fibrillary acidic protein Declarations Consent to Publish declaration All the authors agree for publication if our article is accepted for publication. Date Availability D eclaration The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Consent to Participate declaration: not applicable . Ethics D eclaration All mice were raised by the Laboratory Animal Center, Fujian Medical University following the general guideline of AAALAC handbook and institutional regulations. In accordance with ARRIVE guidelines, the method we used for euthanasia of mice was cervical dislocation. The experimental procedures for the use and care of animals were approved by the Ethics Committee of Fujian Medical University (Permit Number: FMU Ethical Inspection N.2009-32) , China. All experiments were performed in accordance with relevant guidelines and regulations. Competing Interests D eclaration I declare that the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper. Author Contributions D eclaration Q.W. wrote the main manuscript text, analyzed and processed the data and graphs, prepared figures 1,4,5. S.L.prepared figures 2-3. Y.L. prepared figures 6-10, P.L. prepared figures 10, J.H. participated in the processing of the images and data. Corresponding authors C.Z. and Z.H. guided the experiments and the writing of the article. All authors reviewed the manuscript. Q.W., S.L. and Y.L. contributed equally to this work. Acknowledgement We thank Professor Yuan Zhuang and Professor Xiaohui Wu of the Institute of Developmental Biology and Molecular Medicine of Fudan University for providing ADAM10 loxP/loxP transgenic mice for this experiment, the teachers of the Electron Microscope Room of Fujian Medical University for their guidance and assistance, and Professor Fenghua Lan and Dr. Yuqi Zeng of Fujian Medical University for their guidance and help in the experiments. Funding This work was supported by Natural Science Foundation of Fujian Province of China Grant 2010j01171 and Key project of Experimental animal research of Fujian Province of China Grant 2014Y0078. 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(\\u003cstrong\\u003eB)\\u003c/strong\\u003e ADAM10 gene. (1) Cre-ADAM10\\u003csup\\u003eloxP /loxP\\u003c/sup\\u003e transgenic mice. (2) ADAM10\\u003csup\\u003eloxP/loxP\\u003c/sup\\u003e transgenic mice. (3) Cre transgenic mice. M: Marker.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1..png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8193475/v1/8fc56ead82f1b0dabd71b32e.png\"},{\"id\":98436094,\"identity\":\"7eaad41e-825a-4f89-9b3b-74ccee6354e2\",\"added_by\":\"auto\",\"created_at\":\"2025-12-17 16:54:55\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":381670,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eEffects on APP processing of the \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eADAM10 \\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003egene\\u003c/strong\\u003e \\u003cstrong\\u003edeletion\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(\\u003cstrong\\u003eA\\u003c/strong\\u003e)Western blot results. 10% SDS-PAGE was used to separate APP. (\\u003cstrong\\u003eB\\u003c/strong\\u003e) Quantitative statistical analysis of the total content of APP (P \\u0026gt; 0.05), data were not statistically significant. (\\u003cstrong\\u003eC\\u003c/strong\\u003e) Western blot results. 8% SDS-PAGE was used to separate mAPP and imAPP. (\\u003cstrong\\u003eD\\u003c/strong\\u003e,\\u003cstrong\\u003e E\\u003c/strong\\u003e and \\u003cstrong\\u003eF\\u003c/strong\\u003e) Quantitative statistical analysis of mAPP, imAPP and mAPP/imAPP (P \\u0026gt; 0.05), data were not statistically significant. (\\u003cstrong\\u003eG\\u003c/strong\\u003e) Western blot results. 8% SDS-PAGE was used to separate sAPPα and sAPPβ . (\\u003cstrong\\u003eH\\u003c/strong\\u003e) Quantitative statistical analysis of sAPPα and sAPPβ (***P\\u0026lt;0.001, **P\\u0026lt;0.01). (\\u003cstrong\\u003eI\\u003c/strong\\u003e) Western blot results. 16% Tricine-SDS-PAGE was used to separate C99 and C83. (\\u003cstrong\\u003eH\\u003c/strong\\u003e) Quantitative statistical analysis of C99 and C83 (**P \\u0026lt; 0.01, ***P \\u0026lt; 0.001). (\\u003cstrong\\u003eK\\u003c/strong\\u003e)Western blot results. 10% SDS-PAGE was used to separate total Aβ. (\\u003cstrong\\u003eL\\u003c/strong\\u003e) Quantitative statistical analysis of total Aβ (**P \\u0026lt; 0.01, ***P \\u0026lt; 0.001). (\\u003cstrong\\u003eM\\u003c/strong\\u003e) Western blot results. 16% Tricine-SDS-PAGE + 6 M Urea was used to separate Aβ37, Aβ38, Aβ39, Aβ40 and Aβ42/43. (\\u003cstrong\\u003eN\\u003c/strong\\u003e) Quantitative statistical analysis of different lengths of Aβ (P\\u003csup\\u003e37/38/39 \\u003c/sup\\u003e\\u0026gt; 0.05，*P \\u0026lt; 0.05，**P \\u0026lt; 0.01，***P \\u0026lt; 0.001). (control) ADAM10\\u003csup\\u003eloxP/loxP \\u003c/sup\\u003emice. (\\u003cstrong\\u003eO\\u003c/strong\\u003e) The epitope recognition sites of APP-specific antibodies and their binding schematics. (cKO) ADAM10 cKO mice. (Cor. means cortex, Hip. means hippocampus).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8193475/v1/022ce801556195917dd48745.png\"},{\"id\":98437843,\"identity\":\"d5d9bcbf-1e93-4f95-82dc-62eef5e68f74\",\"added_by\":\"auto\",\"created_at\":\"2025-12-17 16:58:07\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":12077,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe concentrations of Aβ40 and Aβ42 in the cortex and hippocampus were detected by ELISA\\u0026nbsp; \\u003c/strong\\u003eResults of Statistical analysis chart (*P \\u0026lt; 0.05，**P \\u0026lt; 0.01，***P \\u0026lt; 0.001). (control)\\u0026nbsp; ADAM10\\u003csup\\u003eloxP/loxP\\u003c/sup\\u003e\\u003csup\\u003e\\u003cem\\u003e \\u003c/em\\u003e\\u003c/sup\\u003emice. (cKO) ADAM10 cKO mice.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8193475/v1/c3b9bda0654bd661f5445280.png\"},{\"id\":98322677,\"identity\":\"e225b7f6-4888-460b-8743-4674887260e8\",\"added_by\":\"auto\",\"created_at\":\"2025-12-16 14:12:12\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":728683,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eImmunohistochemical staining of Aβ in the hippocampus of the brain of mice \\u003c/strong\\u003eFigure \\u003cstrong\\u003eA1\\u003c/strong\\u003e, \\u003cstrong\\u003eB1\\u003c/strong\\u003e, \\u003cstrong\\u003eC1\\u003c/strong\\u003e, \\u003cstrong\\u003eD1\\u003c/strong\\u003e, \\u003cstrong\\u003eE1\\u003c/strong\\u003e, \\u003cstrong\\u003eF1\\u003c/strong\\u003e, \\u003cstrong\\u003eG1\\u003c/strong\\u003e, \\u003cstrong\\u003eH1\\u003c/strong\\u003e, \\u003cstrong\\u003eI1\\u003c/strong\\u003e, \\u003cstrong\\u003eJ1\\u003c/strong\\u003e were taken at 10x, were taken at 10x. \\u003cstrong\\u003eA2\\u003c/strong\\u003e, \\u003cstrong\\u003eB2\\u003c/strong\\u003e, \\u003cstrong\\u003eC2\\u003c/strong\\u003e, \\u003cstrong\\u003eD2\\u003c/strong\\u003e, \\u003cstrong\\u003eE2\\u003c/strong\\u003e, \\u003cstrong\\u003eF2\\u003c/strong\\u003e, \\u003cstrong\\u003eG2\\u003c/strong\\u003e, \\u003cstrong\\u003eH2\\u003c/strong\\u003e, \\u003cstrong\\u003eI2\\u003c/strong\\u003e, \\u003cstrong\\u003eJ2\\u003c/strong\\u003e were taken at 40x, and were higher power views of boxed areas. (MO) Month . (control) ADAM10\\u003csup\\u003eloxP/loxP \\u003c/sup\\u003emice. (cKO) ADAM10 cKO mice.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8193475/v1/64c5f793349006bbe2b524c4.png\"},{\"id\":98436334,\"identity\":\"ec72b725-f1c1-4449-abd4-fc771043c22d\",\"added_by\":\"auto\",\"created_at\":\"2025-12-17 16:55:24\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":715804,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eImmunohistochemical staining of Aβ in the cerebral cortex of mice\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFigure\\u003cstrong\\u003e K1\\u003c/strong\\u003e, \\u003cstrong\\u003eL1\\u003c/strong\\u003e, \\u003cstrong\\u003eN1\\u003c/strong\\u003e, \\u003cstrong\\u003eM1\\u003c/strong\\u003e,\\u003cstrong\\u003e O1\\u003c/strong\\u003e, \\u003cstrong\\u003eP1\\u003c/strong\\u003e, \\u003cstrong\\u003eQ1\\u003c/strong\\u003e, \\u003cstrong\\u003eR1\\u003c/strong\\u003e, \\u003cstrong\\u003eS1\\u003c/strong\\u003e,\\u003cstrong\\u003eT2\\u003c/strong\\u003e were taken at 10x, were taken at 10x. \\u003cstrong\\u003eK2\\u003c/strong\\u003e, \\u003cstrong\\u003eL2\\u003c/strong\\u003e,\\u003cstrong\\u003e N2\\u003c/strong\\u003e,\\u003cstrong\\u003e M2\\u003c/strong\\u003e, \\u003cstrong\\u003eO2\\u003c/strong\\u003e, \\u003cstrong\\u003eP2\\u003c/strong\\u003e, \\u003cstrong\\u003eQ2\\u003c/strong\\u003e, \\u003cstrong\\u003eR2\\u003c/strong\\u003e, \\u003cstrong\\u003eS2\\u003c/strong\\u003e,\\u003cstrong\\u003e T2\\u003c/strong\\u003e were taken at 40x, and were higher power views of boxed areas. (MO) Month. (control) ADAM10\\u003csup\\u003eloxP/loxP \\u003c/sup\\u003emice. (cKO) ADAM10 cKO mice.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8193475/v1/806a5e170372acbf1837a4f1.png\"},{\"id\":98322684,\"identity\":\"c5180c47-444f-47df-aac7-4044cb92257b\",\"added_by\":\"auto\",\"created_at\":\"2025-12-16 14:12:12\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":189397,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eThe levels of Synaptophysin and PSD-95 expression \\u003c/strong\\u003e(\\u003cstrong\\u003eA\\u003c/strong\\u003e) Western blot results of synaptophysin. (\\u003cstrong\\u003eB\\u003c/strong\\u003e) Quantitative statistical analysis of synaptophysin ( *P \\u0026lt; 0.05, **P \\u0026lt; 0.01). (\\u003cstrong\\u003eC\\u003c/strong\\u003e) Western blot results of PSD-95. (\\u003cstrong\\u003eD\\u003c/strong\\u003e) Quantitative statistical analysis of PSD-95 (*P \\u0026lt; 0.05). (control) wild-type C57BL/6 control mice. (cKO) ADAM10 cKO mice.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8193475/v1/e0e93db384078a3d4189d28e.png\"},{\"id\":98437801,\"identity\":\"76a4ad6d-33e0-461b-a945-b7e93f34726d\",\"added_by\":\"auto\",\"created_at\":\"2025-12-17 16:57:50\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2373900,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eImmunohistochemical staining of synaptophysin \\u003c/strong\\u003eSYP antibody (BA3279, PhD, Wuhan) was used to detect synaptophysin. Figure \\u003cstrong\\u003ea\\u003c/strong\\u003e, \\u003cstrong\\u003eb\\u003c/strong\\u003e, \\u003cstrong\\u003ee\\u003c/strong\\u003e, \\u003cstrong\\u003ef\\u003c/strong\\u003e,\\u003cstrong\\u003e i\\u003c/strong\\u003e,\\u003cstrong\\u003e j\\u003c/strong\\u003e,\\u003cstrong\\u003e m\\u003c/strong\\u003e, \\u003cstrong\\u003en\\u003c/strong\\u003e were taken at 10x. Figure \\u003cstrong\\u003ec\\u003c/strong\\u003e, \\u003cstrong\\u003ed\\u003c/strong\\u003e,\\u003cstrong\\u003e g\\u003c/strong\\u003e, \\u003cstrong\\u003eh\\u003c/strong\\u003e,\\u003cstrong\\u003e k\\u003c/strong\\u003e,\\u003cstrong\\u003el\\u003c/strong\\u003e, \\u003cstrong\\u003eo\\u003c/strong\\u003e, \\u003cstrong\\u003ep\\u003c/strong\\u003e were taken at 40x, and were higher power views of boxed areas. (control) wild-type C57BL/6 control mice. (cKO) \\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003eADAM10 cKO mice.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8193475/v1/74c0709acca2167de4f32f6a.png\"},{\"id\":98322681,\"identity\":\"16b8d2d6-6ee6-483c-a071-caca5179773b\",\"added_by\":\"auto\",\"created_at\":\"2025-12-16 14:12:12\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1307603,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eImmunohistochemical staining of PSD-95 \\u0026nbsp;\\u003c/strong\\u003ePSD-95 antibody (Thermo Fisher, MA1-045) was used to detect PSD-95. Figure \\u003cstrong\\u003ea\\u003c/strong\\u003e, \\u003cstrong\\u003eb\\u003c/strong\\u003e, \\u003cstrong\\u003ee\\u003c/strong\\u003e,\\u003cstrong\\u003ef\\u003c/strong\\u003e,\\u003cstrong\\u003e i\\u003c/strong\\u003e,\\u003cstrong\\u003e j\\u003c/strong\\u003e were taken at 10x. Figure \\u003cstrong\\u003ec\\u003c/strong\\u003e, \\u003cstrong\\u003ed\\u003c/strong\\u003e,\\u003cstrong\\u003e g\\u003c/strong\\u003e, \\u003cstrong\\u003eh\\u003c/strong\\u003e,\\u003cstrong\\u003e k\\u003c/strong\\u003e,\\u003cstrong\\u003e l\\u003c/strong\\u003e were taken at 40x, and were higher power views of boxed areas. (control) wild-type C57BL/6 control mice. (cKO) ADAM10 cKO mice.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8193475/v1/f92dd5e4a259fc10723f67a8.png\"},{\"id\":98437698,\"identity\":\"e11b21f3-f834-454e-ad3e-ee9aa3f3d41d\",\"added_by\":\"auto\",\"created_at\":\"2025-12-17 16:57:34\",\"extension\":\"png\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":11517,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eExpression of synaptophysin and PSD-95 in hippocampus and cortex of the two groups of mice(mean optical density)(x±s) \\u003c/strong\\u003e(\\u003cstrong\\u003eA\\u003c/strong\\u003e) Quantitative statistical analysis mean optical density of synaptophysin(*P \\u0026lt; 0.05，**P \\u0026lt; 0.01，***P \\u0026lt; 0.001).(\\u003cstrong\\u003eB\\u003c/strong\\u003e)Quantitative statistical analysis mean optical density of PSD-95 (*P \\u0026lt; 0.05). (control) wild-type C57BL/6 control mice. (cKO) ADAM10 cKO mice.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure9.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8193475/v1/99f60c3623b3be8b71967df4.png\"},{\"id\":98436577,\"identity\":\"aeb71097-3834-4ea4-9ba3-a6a52d6206bf\",\"added_by\":\"auto\",\"created_at\":\"2025-12-17 16:55:55\",\"extension\":\"png\",\"order_by\":10,\"title\":\"Figure 10\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":767461,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSynaptic ultrastructure and LTP experiment \\u003c/strong\\u003e(\\u003cstrong\\u003eA\\u003c/strong\\u003e) The synaptic ultrastructure of C57BL/6 control mice (\\u003cstrong\\u003eB\\u003c/strong\\u003e) The synaptic ultrastructure of \\u003cem\\u003eADAM10 \\u003c/em\\u003ecKO mice. Electron microscopy, 25000X, → excitatory synapses.\\u003c/p\\u003e\\n\\u003cp\\u003e(\\u003cstrong\\u003eC\\u003c/strong\\u003e) Original recording of LTP brain potentials in mice. (\\u003cstrong\\u003eD\\u003c/strong\\u003e) LTP field potential slope analysis scatter plot. (\\u003cstrong\\u003eE\\u003c/strong\\u003e) LTP field potential amplitude analysis scatter plot. (control) wild-type C57BL/6 control mice. (cKO) ADAM10 cKO mice.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure10.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8193475/v1/0b2efcd7884eabe437c6af09.png\"},{\"id\":98435816,\"identity\":\"65064fe9-55f9-4210-8b8a-5fd1843b30e0\",\"added_by\":\"auto\",\"created_at\":\"2025-12-17 16:54:27\",\"extension\":\"png\",\"order_by\":11,\"title\":\"Figure 11\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1610724,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eImmunofluorescence was used to detect GFAP\\u003c/strong\\u003e \\u0026nbsp;Immunofluorescence images of the mouse brain hippocampus and cortex: (\\u003cstrong\\u003ea\\u003c/strong\\u003e) Control DAPI staining, (\\u003cstrong\\u003eb\\u003c/strong\\u003e) Control GFAP immunofluorescence, (\\u003cstrong\\u003ec\\u003c/strong\\u003e) Control confocal merge; (\\u003cstrong\\u003ed\\u003c/strong\\u003e) cKO DAPI staining, (e) cKO GFAP immunofluorescence, (f) cKO confocal merge.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure11.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8193475/v1/bb0cddecfbf96e1d0ae9bc64.png\"},{\"id\":105561828,\"identity\":\"cd3d2c0d-8b80-47ac-bb5d-4b30a3f755d3\",\"added_by\":\"auto\",\"created_at\":\"2026-03-27 12:12:16\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":11981225,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8193475/v1/7a26deef-9d13-4d9c-b480-b3a34de4f86f.pdf\"},{\"id\":98437845,\"identity\":\"e8b8ff78-d334-4957-b480-2250ab7bd926\",\"added_by\":\"auto\",\"created_at\":\"2025-12-17 16:58:07\",\"extension\":\"7z\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":112930,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"rawdata.7z\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8193475/v1/fc7130e9a021a0d64eaccbf1.7z\"},{\"id\":98437008,\"identity\":\"764fb967-b832-4a03-82eb-94b1a51d7566\",\"added_by\":\"auto\",\"created_at\":\"2025-12-17 16:56:43\",\"extension\":\"pdf\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":2841298,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"thefulluncroppedGelsandBlotsimages.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8193475/v1/e357d9d0a3268a126318e52b.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Effects of ADAM10 deletion on APP shedding, synapse and synaptic plasticity in adult mice\",\"fulltext\":[{\"header\":\"1 INTRODUCTION\",\"content\":\"\\u003cp\\u003eAlzheimer's Disease (AD), which mainly occurs in individuals aged 65 or older, is one of the most common neurodegenerative diseases. This disease is one of the sixth leading causes of death worldwide, and its incidence continues to rise annually, creating a heavy burden for society and families [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. The amyloid precursor protein (APP), a type I transmembrane protein associated with AD, primarily mediates physiological and pathological processes through proteolytic cleavage fragments, including the amyloid-beta (Aβ) peptide [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. Amyloid plaques, one of the hallmark, pathological features in the brain tissue of AD patients, and are predominantly composed of Aβ peptides [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e][\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. Aβ peptide is generated by the APP through the amyloidogenic cleavage pathway. In the amyloidogenic cleavage pathway, the ectodomain shedding of the APP ,which is firstly done by β -secretase, generates the soluble APPβ (sAPPβ) and the C-terminal fragment 99 (C99). Subsequently, the cleavage of C99 generates Aβ peptides of varying lengths (38\\u0026ndash;49 amino acids) and APP intracellular domains (AICD) by γ-secretase. However, another proteolytic processing pathway exists for APP. In this pathway, APP shedding is firstly done by α-secretase but not by β-secretase, and the site of hydrolysis is within Aβ peptide (at the lysine16-leucine17 bond), generating the soluble APPα (sAPPα) and the C-terminal fragment 83 (C83). Then, the cleavage of C83 releases small P3 fragment and AICD by γ-secretase. Thereby APP cleavage by α-secretase can prevent Aβ formation [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e][\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e][\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. It has now been confirmed that amyloid plaques do not contain P3, and sAPPα has neurotrophic and neuroprotective effects [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e][\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e][\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eDisntegrin/metalloproteinase 10 (\\u003cem\\u003eADAM10\\u003c/em\\u003e), a member of the \\u003cem\\u003eADAMs\\u003c/em\\u003e (a deintegrin and metalloproteinases) family, is widely expressed in mammalian cells and mediates the hydrolysis of various cellular receptors and signaling molecules (e.g., APP and Notch) [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. ADAM10 has been identified as the principal constitutive α-secretase responsible for APP processing in neuronal cells. Notably, downregulation of ADAM10 gene expression in primary cultured neurons significantly reduces sAPPα production, while enhancing sAPPβ and Aβ igeneration [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. However, a study by Jorissen E et al. demonstrated that neuronal ADAM10 deletion in neural precursor cell conditional knockout mice not only decreases C83 and sAPPα, but also paradoxically reduces sAPPβ, C99, total Aβ, Aβ40, and Aβ42 [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. Intriguingly, additional evidence suggests that α-secretase and β-secretase activities are not strictly interdependent in primary cortical neurons. Specifically, inhibition of β-secretase activity could improve the activity of α-secretase, but inhibition of α-secretase activity does not improve the activity of APP cleavage by β-secretase [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eAll data mentioned above come from in vitro studies. However, a recent in vivo study showed that ADAM10 gene expression is significantly downregulated in the cerebral cortex of ADAM10 cKO mice, leading to reduced sAPPα and elevated sAPPβ and endogenous Aβ production [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. These findings suggest that α-secretase competes with β-secretase for APP cleavage in vivo. Consequently, enhancing α-secretase activity could not only suppress Aβ generation but also promote sAPPα production, potentially offering a dual therapeutic strategy for treating or even preventing AD.\\u003c/p\\u003e \\u003cp\\u003eSynapses are specialized intercellular junctions between neurons or between neurons and non-nerve cells (e.g.,muscle cells, gland cells). They serve as critical sites for both neuronal connectivity and information transmission. Generally the presynaptic terminal typically exhibits spherical swelling containing numerous synaptic vesicles and mitochondrion. The thickened axonal membrane forms the presynaptic membrane, which harbors voltage-gated, calcium ion channels, and membrane fusion proteins (e.g., SNAP25, Syntaxin). Synaptic cleft is located between the pre- and postsynaptic membranes, it contains various adhesion molecules essential for signal transduction. Synapses are classified into type I and type Ⅱ based on differences in the electron-dense band thickness of the pre- and postsynaptic membranes. The type I synapses (excitatory synapses) exhibit asymmetric pre- and postsynaptic membranes, with the postsynaptic membrane being thicker than the presynaptic membrane. The type Ⅱ synapses (inhibitory synapses) characterized by symmetrical pre- and postsynaptic membranes of comparable thickness. Synaptic plasticity is that synapses dynamically adapt their functional efficiency (e.g., long-term potentiation [LTP] or depression [LTD]) and morphology (e.g., postsynaptic density [PSD] thickening, synaptic cleft narrowing) in response to activity. For example, tetanic stimulation of hippocampal synapses induces LTP-a sustained increase in excitatory postsynaptic potential (EPSP) amplitude།which is pivotal for studying cognitive memory mechanisms [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. Many neurodegenerative diseases show pathological features of synapses, including abnormal expression of synaptic proteins, synaptic loss, synaptic dysfunction, and structural alterations of synapses [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e][\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eTherefore, we used the ADAM10 conditional knockout (cKO) mouse model established by our research group to investigate the effects of ADAM10 gene on APP metabolism [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e], aiming to elucidate the interplay the relationship between ADAM10 α-secretase and β-secretase in the substrate APP cleavage in vivo. Additionally, we examined the synaptic-related protein, synaptic ultrastructure and hippocampus LTP in ADAM10 cKO mice to further explore the effects of ADAM10 gene on synaptic function in adult mice. These findings advance our understanding of the ADAM10\\u0026rsquo;s physiological and pathological functions.\\u003c/p\\u003e\"},{\"header\":\"2 MATERIALS AND METHODS\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\"\\u003e\\n \\u003ch2\\u003e2.1 MATERIALS\\u003c/h2\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAntibodies and Kits\\u003c/strong\\u003e Antibodies used for Western blot were shown in Table\\u0026nbsp;2. HRP AffiniPure goat anti-rabbit IgG (H\\u0026thinsp;+\\u0026thinsp;L) ( E030120 ) was purchased from EarthOx, LLC\\u0026middot;San Francisco, CA\\u0026middot;USA. HRP AffiniPure goat anti-mouse IgG (H\\u0026thinsp;+\\u0026thinsp;L) (EM35110) was purchased from Beijing Emarbio Science \\u0026amp; Technology Co.,LTD.. Mouse A\\u0026beta;40 ELISA Kit (E-EL-M0067c) and mouse A\\u0026beta;42 ELISA kit (E-EL-M0068c) were purchased from Elabscience. The mouse monoclonal GFAP antibody (CBL412C3) was purchased from Millipore. An anti-mouse Cy3-conjugated secondary antibody kit was purchased from Beyotime. High-sensitivity ECL chemiluminescence kit was purchased from Heron Bio Inc. SABC immunohistochemical staining kit (SA1021) was purchased from Wuhan Doctor De Bio Co., LTD.. Mouse high-sensitivity two-step immunohistochemical detection kit (PV9002) was purchased from Zhongshan Jinqiao Biological Co., LTD.. DAB kit was purchased from Beijing Zhuang Meng International Biological Gene Technology Co.,LTD.. BCA kit (P0012S) was purchased from Beyotime. PCR primers were synthesized by Shanghai Sangon Biological Engineering Technology Service Co., LTD..\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cimg 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\\\"\\u003e\\u003c/p\\u003e\\n \\u003cdiv\\u003e\\n \\u003ctable id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv\\u003eTable 2\\u003c/div\\u003e\\n \\u003cdiv\\u003e\\n \\u003cp\\u003eA list of antibodies were used for Western blot.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eTarget\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eAntibody(Catalog No.)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ecompany\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eTotal A\\u0026beta;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eAnti-\\u0026beta;-Amyloid antibody(B-4,sc-28365)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eSanta Cruz Biotechnology\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eTotal FL-APP,total sAPP, mAPP, imAPP, sAPP\\u0026alpha; and sAPP\\u0026beta;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eAnti-Alzheimer Precursor Protein A4, a.a 66\\u0026ndash;81 of APP antibody(22C11)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eMillipore\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eC99,C83\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eAnti-Amyloid Precursor Protein C-terminal antibody(A8718)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eSigma\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eA\\u0026beta;37, A\\u0026beta;38, A\\u0026beta;39, A\\u0026beta;40 and A\\u0026beta;42/43\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u0026beta;-Amyloid, 1\\u0026ndash;16 Monoclonal Antibody(6E10, SIG-39320)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eCovance\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u0026beta;-actin\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eAnti-\\u0026beta;-actin Mouse Monoclonal antibody(E021020)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eEarthOx, LLC\\u0026middot;San Francisco, CA\\u0026middot;USA\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003esynaptophysin\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eAnti-synaptophysin Antibody(SC-17750)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eSanta Cruz Biotechnology\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePSD-95\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eAnti-PSD-95 Antibody(MA1-045)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eThermo Fisher\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u0026beta;-actin\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eAnti-\\u0026beta;-actin (MJ3007)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eearth\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n \\u003c/div\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eExperimental Animals\\u003c/strong\\u003e ADAM10\\u003csup\\u003eloxP/loxP\\u003c/sup\\u003e transgenic mice were provided by Professors Yuan Zhuang and Xiaohui Wu, Institute of Developmental Biology and Molecular Medicine, Fudan University. CAMKII\\u0026alpha;-Cre transgenic mice(on a C57BL/6 pure background) were prepared and identified by our research group. Clean grade C57BL/6J mice were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences. All experimental mice were raised by the Laboratory Animal Center, Fujian Medical University. In accordance with ARRIVE guidelines, the method we used for euthanasia of mice was cervical dislocation. The experimental procedures for the use and care of animals were approved by the Ethics Committee of Fujian Medical University, China. All experiments were performed in accordance with relevant guidelines and regulations.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec4\\\"\\u003e\\n \\u003ch2\\u003e2.2 METHODS\\u003c/h2\\u003e\\n \\u003cdiv id=\\\"Sec5\\\"\\u003e\\n \\u003ch2\\u003e2.2.1 Generation and genotype analysis of ADAM10 cKO mice\\u003c/h2\\u003e\\n \\u003cp\\u003eCaMKⅡ\\u0026alpha;-Cre/ADAM10\\u003cstrong\\u003e-/\\u003c/strong\\u003e+ mice were generated by crossing \\u003cem\\u003eCaMKⅡ\\u0026alpha;-Cre\\u003c/em\\u003e transgenic mice with ADAM10\\u003csup\\u003eloxP/loxP\\u003c/sup\\u003e transgenic mice. Subsequently, ADAM10 cKO mice were obtained by breeding CaMKII\\u0026alpha;-Cre/ADAM10\\u003cstrong\\u003e-/\\u003c/strong\\u003e+mice with ADAM10\\u003csup\\u003eloxP/loxP\\u003c/sup\\u003e transgenic mice. Tail DNA Extraction: Genomic DNA was isolated from mice 10\\u0026ndash;14 days postnatally. PCR Amplification: DNA samples were amplified using primers F1/R1 and KOF/EKOB (see Table 1). Electrophoresis: Mouse genotypes were confirmed by agarose gel electrophoresis. Control groups: ADAM10\\u003csup\\u003eloxP/loxP\\u003c/sup\\u003e \\u003cem\\u003emice\\u003c/em\\u003e, or wild-type C57BL/6J mice.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003cdiv id=\\\"Sec6\\\"\\u003e\\n \\u003ch2\\u003e2.2.2 Western blotting\\u003c/h2\\u003e\\n \\u003cp\\u003eADAM10 cKO mice, ADAM10\\u003csup\\u003eloxP/loxP\\u003c/sup\\u003e mice and wild-type C57BL/6J mice were selected for Western blot analysis. The mice were euthanized via cervical dislocation, and then whole brains were rapidly dissected. The cortex, hippocampus and cerebellum were isolated from the brain. Then total protein was extracted using a lysis buffer. Protein concentrations were quantified using a BCA kit (P0010s, Biyuntian Biotechnology Co., LTD.). Protein (20\\u0026ndash;60\\u0026micro;g per lane) were separated on 8% SDS-PAGE gels, 10% SDS-PAGE gels, 16% Tricine-SDS-PAGE gels, and 16% Tricine-SDS-PAGE\\u0026thinsp;+\\u0026thinsp;6M Urea gels, and transferred to polyvinylidene difluoride (PVDF, Bio-Rad) membranes for western blot analysis. Band intensities were quantified using ImageJ software (National Institutes of Health). .Statistical comparisons between groups were performed using Student\\u0026rsquo;s t-test.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003cdiv id=\\\"Sec7\\\"\\u003e\\n \\u003ch2\\u003e2.2.3 ELISA(enzyme-linked immunosorbent assay)\\u003c/h2\\u003e\\n \\u003cp\\u003eProteins were extracted from the forebrain cortex and hippocampus of twelve-month-old ADAM10 cKO mice and ADAM10\\u003csup\\u003eloxP/loxP\\u003c/sup\\u003e control mice, followed by Western blot analysis. Total protein concentration was determined using a BCA kit (P0010s, Biyuntian Biotechnology Co., LTD.). We used ELISA to detect the concentrations of A\\u0026beta;40 and A\\u0026beta;42 in the extracted proteins. The Mouse A\\u0026beta;40/A\\u0026beta;42 ELISA Kits (Elabscience) employ a double antibody sandwich method. Experiments were performed according to the manufacturer\\u0026rsquo;s instructions. Optical density (OD) values were measured at 450nm using a microplate reader, A\\u0026beta;40/A\\u0026beta;42 concentrations were directly proportional to the OD 450 values, and calculated by plotting standard curves.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003cdiv id=\\\"Sec8\\\"\\u003e\\n \\u003ch2\\u003e2.2.4 Immunohistochemistry\\u003c/h2\\u003e\\n \\u003cp\\u003eTwelve-month-old ADAM10 cKO mice, ADAM10\\u003csup\\u003eloxP/loxP\\u003c/sup\\u003e control mice and wild-type C57BL/6J control mice were were used for these experiments. The mice were euthanized via cervical dislocation, the brains were rapidly dissected and fixed in 4% paraformaldehyde for 24 hours. Brain tissues were Paraffin-embedded, and 5 \\u0026micro;m-thick coronal sections were prepared. We performed immunohistochemical staining. For A\\u0026beta; staining, we used a SABC kit, a mouse anti-\\u0026beta;-amyloid antibody(B-4) and a goat anti-mouse secondary antibody. Sections were developed with 3,3\\u0026apos;-diaminobenzidine (DAB) and counterstained with hematoxylin. For synaptophysin staining, we used a SABC kit, an anti-synaptophysin antibody (SC-17750) and a goat anti-mouse secondary antibody. For PSD-95 staining, we used a high-sensitivity two-step kit and an anti-PSD-95 antibody (MA1-045). After dehydration and xylene-based clearing, sections were sealed with neutral resin and dried at 37℃. Images were acquired using a bright-field microscope, and mean optical density was quantified with Image-Pro Plus 6.0.We compared the differences in protein expression between the two groups.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003cdiv id=\\\"Sec9\\\"\\u003e\\n \\u003ch2\\u003e2.2.5 Synaptic ultrastructure\\u003c/h2\\u003e\\n \\u003cp\\u003eWe used transmission electron microscopy(TEM) to compare ultrastructure differences between experimental and control mice. Twelve-month-old ADAM10 cKO mice and wild-type C57BL/6J control mice were used in this study. The mice were euthanized via cervical dislocation and their skulls were opened to expose the brain. 4% paraformaldehyde solution was immediately applied to the brain tissue, which was rapidly dissected isolate the hippocampal CA1 regio. Tissue blocks from the hippocampal CA1 region were trimmed to approximately 1 \\u0026times; 1 \\u0026times; 1 mm\\u0026sup3; for TEM processing. The tissue blocks were pre-fixed with 3% glutaraldehyde and 1.5% paraformaldehyde in 0.1 M PBS (pH7.2) for \\u0026ge;\\u0026thinsp;2 hours at 4℃, followed by three washes with 0.1 M PBS (pH7.2). Post-fixation was performed using 1% osmium tetroxide and 1.5% potassium ferrocyanide in PBS for 1.5 hours at 4\\u0026deg;C, after which the tissues were rinsed three times with PBS. The samples were then dehydrated through a graded ethanol series, infiltrated, and embedded in Epoxy Resin 618. Ultrathin sections (100 nm thickness) were prepared, double-stained with uranyl acetate and lead citrate, and imaged using a Philips 208 TEM. Excitatory synapses were identified based on established ultrastructural criteria[18]: a thickened postsynaptic density (PSD), presence of densely stained bands in the postsynaptic membrane, and spherical synaptic vesicles. For quantitative analysis, PSD thickness and synaptic cleft width were measured using stereological methods with square test grids (side length: 86.67 nm) Synaptic interface curvature was calculated as the ratio of the arc length (A) to the chord length (B) of the synaptic membrane. All measurements were analyzed using Image-Pro Plus 6.0 software.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003cdiv id=\\\"Sec10\\\"\\u003e\\n \\u003ch2\\u003e2.2.6 Hippocampal LTP experiment\\u003c/h2\\u003e\\n \\u003cp\\u003eThe nine-month-old ADAM10 cKO mice and wild-type C57BL/6J control mice were used for electrophysiological experiments. Hippocampal brain slices (400 \\u0026micro;m thick) were prepared according to experimental protocols. The slices were incubated for 2\\u0026ndash;3 hours prior to recording. For recordings, a single incubated slice was transferred to a semi-immersion thermostatic bath (32\\u0026deg;C) on a nylon mesh. A 95% O₂/5% CO₂ gas mixture was continuously perfused above the slice, while non-oxygenated artificial cerebrospinal fluid (ACSF) was irrigated beneath it at a constant rate (1\\u0026ndash;2 ml/min). Under an operating microscope, a bipolar tungsten stimulation electrode (bare tip) was positioned on the Schaffer collateral pathway in the hippocampal CA3 region. The electrode was connected to a stimulator via an isolator, with stimulation parameters set to 100\\u0026ndash;300 \\u0026micro;s pulse width, 0.1\\u0026ndash;0.6 mA intensity, and 2\\u0026ndash;10 s interstimulus intervals. A recording electrode was placed in the CA1 pyramidal cell layer to measure evoked potential. Signals were amplified using a microelectrode amplifier, then routed to a memory oscilloscope, a computer-based data acquisition system, and analytical software.This setup allowed real-time visualization of evoked potentials on both the oscilloscope and computer for simultaneous recording and analysis.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003cdiv id=\\\"Sec11\\\"\\u003e\\n \\u003ch2\\u003e2.2.7 Immunofluorescence detection of glial fibrillary acidic protein (GFAP) expression\\u003c/h2\\u003e\\n \\u003cp\\u003eTwelve-month-old ADAM10 cKO mice and wild-type C57BL/6J control mice were were used for these experiments. The mice were euthanized via cervical dislocation, the brains were rapidly dissected and fixed in 4% paraformaldehyde for 24 hours. 5 \\u0026micro;m-thick frozen sections were prepared. For immunofluorescence staining, we incubated the brain sections with a mouse monoclonal GFAP antibody (Millipore) followed by a fluorophore-conjugated secondary antibody. Nuclei were counterstained with DAPI. The slides were mounted using anti-fade mounting mediumto prevent fluorescence quenching. All procedures were performed under minimal light exposure to avoid photobleaching. Confocal microscopy was used for imaging and analysis. By comparing the fluorescence intensity between the experimental (cKO) and control groups, we could directly assess GFAP expression levels.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003cdiv id=\\\"Sec12\\\"\\u003e\\n \\u003ch2\\u003e2.2.8 Data processing\\u003c/h2\\u003e\\n \\u003cp\\u003eStatistical significance was calculated by Student\\u0026rsquo;s t-test using SPSS17.0 software. Data were expressed as (\\u0026plusmn;\\u0026thinsp;s), *P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, **P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01, ***P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"3 Results\",\"content\":\"\\u003cdiv id=\\\"Sec14\\\"\\u003e\\n \\u003ch2\\u003e3.1 Generation genotype analysis of ADAM10 cKO mice\\u003c/h2\\u003e\\n \\u003cp\\u003eGenotype analysis of progeny mice by gel electrophoresis revealed distinct amplification patterns (see Fig.\\u0026nbsp;1). In Cre-transgenic mice, the amplified Cre gene fragment measured 975 bp, whereas wild-type controls showed no detectable bands (see Fig.\\u0026nbsp;1A). Wild-type ADAM10 alleles produced 1,200-bp bands, while ADAM10 alleles with LoxP markers generated 1,000-bp bands (see Fig.\\u0026nbsp;1B).. Mice harboring both \\u003cem\\u003eADAM10\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eloxP/loxP\\u003c/em\\u003e\\u003c/sup\\u003e and Cre genes were selected for subsequent experiments.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec15\\\"\\u003e\\n \\u003ch2\\u003e3.2 Effects on APP processing of deletion of the ADAM10 gene\\u003c/h2\\u003e\\n \\u003cp\\u003eTwelve-month-old ADAM10 cKO mice and ADAM10\\u003csup\\u003eloxP/lox\\u003cem\\u003eP\\u003c/em\\u003e\\u003c/sup\\u003e control mice were analyzed. The epitope recognition sites of APP-specific antibodies and their binding schematics are illustrated in Fig.\\u0026nbsp;2O. For the protein amount, the target protein-to-β-actin ratios were included in statistical analyses to control for intra-group variations in sample loading. The results showed that compared with the controls, sAPPα levels decreased by 8.38% (P \\u0026lt; 0.001, n = 4 per group) and 10.15% (P \\u0026lt; 0.01) in the cortex and hippocampus, respectively(see Fig.\\u0026nbsp;2G-H); C83 levels declined by 13.98% (P \\u0026lt; 0.01, n = 4 per group) and 18.93% (P \\u0026lt; 0.01) in the cortex and hippocampus, respectively (see Fig.\\u0026nbsp;2I-J); sAPPβ levels increased by 10.31% (P \\u0026lt; 0.05, n = 4 per group) and 12.54% (P \\u0026lt; 0.001) in the cortex and hippocampus, respectively (see Fig.\\u0026nbsp;2G-H); C99 levels increased by 16.64% (P \\u0026lt; 0.01, n = 4 per group) and 21.55% (P \\u0026lt; 0.001) in the cortex and hippocampus, respectively(see Fig.\\u0026nbsp;2I-J); and total Aβ levels increased by 30.98% (P \\u0026lt; 0.01, n = 4 per group) and 58.97% (P \\u0026lt; 0.001) in the cortex and hippocampus, respectively(see Fig.\\u0026nbsp;2K-L). However, total full-length APP (total FL-APP), total soluble APP (total sAPP), mature APP (mAPP), and immature APP (imAPP) were not different between the two groups in the cerebral cortex and hippocampus (see Fig.\\u0026nbsp;2A-F). We used 16% Tricine-SDS-PAGE + 6 M Urea gels to isolate Aβ peptides of different lengths. The results showed that compared with the controls, Aβ40 levels in the cortex and hippocampus of ADAM10 cKO mice increased by 9.60% (P \\u0026lt; 0.05, n = 4 per group) and 17.91% (P \\u0026lt; 0.01), respectively; Aβ42/43 increased by 31.70% (P \\u0026lt; 0.01, n = 4 per group) and 76.93% (P \\u0026lt; 0.001) in the cortex and hippocampus of ADAM10 cKO mice, respectively. There was no significant difference in Aβ37, Aβ38, or Aβ39 between the two groups (see Fig.\\u0026nbsp;2M-N).\\u003c/p\\u003e\\n \\u003cp\\u003eWe utilized ELISA to quantify Aβ40 and Aβ42 levels in the cerebral cortex and hippocampus of ADAM10 cKO mice and ADAM10\\u003csup\\u003eloxP/loxP\\u003c/sup\\u003e control mice. The experimental results showed that compared with the controls, Aβ40 levels in the cortex and hippocampus of ADAM10 cKO mice increased by 17.91% (P \\u0026lt; 0.05, n = 4 per group) and 39.53% (P \\u0026lt; 0.01), respectively. Similarly, Aβ42 in the cerebral cortex and hippocampus increased by 31.70% (P \\u0026lt; 0.01, n = 4 per group) and 76.93% (P \\u0026lt; 0.001), respectively (see Fig.\\u0026nbsp;3). These ELISA findings were consistent with the Western blotting results.\\u003c/p\\u003e\\n \\u003cp\\u003eImmunohistochemistry was employed to assess amyloid plaque deposition in ADAM10 cKO mouse brains. Brain sections from \\u003cem\\u003eADAM10\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eloxP/loxP\\u003c/em\\u003e\\u003c/sup\\u003e control mice and ADAM10 cKO mice at 7-,9-,11-, 13-, and 15-month-old (three pairs per group) were stained with mouse anti-β-amyloid monoclonal antibody (B-4) to detect Aβ peptides in the cerebral cortex and hippocampus.The results demonstrated that neither transgenic mouse strain exhibited amyloid plaque deposition. However, soluble Aβ peptides were observed within neuronal cells of both groups.Notably, compared to controls, ADAM10 cKO mice displayed more intense staining of Aβ products in cortical and hippocampal neurons, with a pronounced age-dependent progression in staining intensity (see Fig.\\u0026nbsp;4, Fig.\\u0026nbsp;5)。\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec16\\\"\\u003e\\n \\u003ch2\\u003e3.3 The expressions of synaptophysin and PSD-95 were analyzed by Western blot and immunohistochemistry\\u003c/h2\\u003e\\n \\u003cp\\u003eWestern blot was performed to detect synaptophysin and PSD-95 levels in the cortex and hippocampus. For the protein amount, the target protein-to-β-actin ratios were included in statistical analyses to control for intra-group variations in sample loading. The results showed that, compared with the controls, synaptophysin levels in cortex and hippocampus of \\u003cem\\u003eADAM10\\u003c/em\\u003e cKO mice significantly reduced by 24.75% (P \\u0026lt; 0.05, n = 3 per group) and 40.10% (P \\u0026lt; 0.01), respectively(see Fig.\\u0026nbsp;6A-B). In contrast, PSD-95 levels showed a 23.37% decrease in hippocampus of \\u003cem\\u003eADAM10\\u003c/em\\u003e cKO mice (P \\u0026lt; 0.05, n = 3 per group), with no significant differences observed incortical or cerebellar PSD-95 levels between the two groups (see Fig.\\u0026nbsp;6C-D).\\u003c/p\\u003e\\n \\u003cp\\u003eImmunohistochemical results showed that the synaptophysin was a brown-yellow granular, punctate, or linear immunoreactive deposits with lamellar distributions in hippocampal CA1, CA3, gyrus (DG), and cerebral cortex. Compared with the controls, the experimental groups exhibited synaptophysin immunoreactivity with reduced density, pale staining, and diminishied structures (see Fig.\\u0026nbsp;7). MOD analysis demonstrated significant decreases in synaptophysin levels in ADAM10 cKO mice: 18.78% reduction in CA1 (P \\u0026lt; 0.001, n = 3 per group), 14.00% in CA3 (P \\u0026lt; 0.01), 21.17% in DG (P \\u0026lt; 0.01), and 24.59% reduction in cerebral cortex (P \\u0026lt; 0.05) (see Fig.\\u0026nbsp;9A). PSD-95 immunoreactivity presented as brown-yellow granular deposits in the hippocampus CA1, CA3 and the cerebral cortex (see Fig.\\u0026nbsp;8). In experimental groups, CA1 showed lighter-stained PSD-95 granules compared to controls. MOD analysis indicated 26.23% decrease in hippocampal CA1 PSD-95 content (P \\u0026lt; 0.05, n = 3 per group), whereas cortical and CA3 regions showed no significant differences (P \\u0026gt; 0.05, n = 3 per group) (see Fig.\\u0026nbsp;9B). These findings were consistent with previous Western blot results.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec17\\\"\\u003e\\n \\u003ch2\\u003e3.4 Synaptic ultrastructure and hippocampal LTP\\u003c/h2\\u003e\\n \\u003cp\\u003eThe ultrastructure of synapses is shown in Fig.\\u0026nbsp;10A-B. In the synaptic ultrastructure, mitochondria and numerous spherical synaptic vesicles can be observed in the presynaptic region. Clearly distinguishable presynaptic and postsynaptic membranes, along with the synaptic cleft, are visible. In some synapses, the postsynaptic membrane is noticeably thicker than the presynaptic membrane. Quantitative analysis revealed that excitatory synapses in ADAM10 cKO mice displayed significantly thinner PSD (P \\u0026lt; 0.05), reduced synaptic interface curvature (P \\u0026lt; 0.05), and widened synaptic clefts (P \\u0026lt; 0.05) compared to wild-type C57BL/6J controls (n = 3 per group, see Table\\u0026nbsp;3). These findings indicate that ADAM10 deletion induces synaptic ultrastructural abnormalities and impairs synaptic plasticity in hippocampal neurons of ADAM10 cKO mice.\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cimg 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\\\"\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eThe hippocampal LTP experiment serves as a key physiological correlate of synaptic plasticity, providing mechanistic insights into behavioral regulation. The experimental results shown that baseline LTP was successfully induced in both groups prior to high-frequency stimulation (HFS). Following HFS, ADAM10 cKO mice exhibited significantly reduced field potential slopes and amplitudes. These findings revealed that LTP induction was significantly inhibited after 30min(see Fig. 10C-E).\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec18\\\"\\u003e\\n \\u003ch2\\u003e3.5 The expression of GFAP gene in the cerebral cortex and hippocampus of ADAM10 cKO mice\\u003c/h2\\u003e\\n \\u003cp\\u003eWe selected 12-month-old ADAM10 cKO mice and wild-type C57BL/6 control mice, using GFAP-specific labeling to mark radial glial cells and astrocytes in the mouse brain. The results are shown in Fig.\\u0026nbsp;11. Compared with the control group, the GFAP fluorescence intensity was enhanced in the cerebral cortex and hippocampus of ADAM10 cKO mice. These experimental results indicate that after the deletion of the ADAM10 gene, GFAP expression increased in the cerebral cortex and hippocampus of the mice, accompanied by astrocyte proliferation.\\u003c/p\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"4 DISCUSSION\",\"content\":\"\\u003cp\\u003eWe used our ADAM10 cKO mouse model to investigate the function of ADAM10 in adult mice. ADAM10 gene deletion in the forebrain cortex and hippocampus, with higher knockout efficiency in the hippocampus compared to the cortex, demonstrating spatial specificity [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. Johannes Prox et al. previously generated a brain-specific ADAM10 cKO model using the CaMKⅡα-Cre promoter[\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e], which we also employed. However,phenotypic differences (e.g., survival rate, life span, epilepsy incidence) exist between the two models, likely due to variations in Cre gene copy number, genomic insertion site, and other undefined factors. Notably, Our ADAM10 cKO mice exhibit significantly lower epilepsy incidence and mortality, along with extended lifespans (some surviving\\u0026thinsp;\\u0026gt;\\u0026thinsp;2 years), compared to the Prox et al. model. In our model, epilepsy does not manifest post-weaning and typically emerges after 10 months of age. Initial seizures are brief and sporadic, but frequency and duration increase progressively with age. These phenotypic advantages make our ADAM10 cKO model particularly suitable for studying Alzheimer\\u0026rsquo;s disease pathogenesis, as chronic neurodegeneration studies require prolonged survival. So, we used our ADAM10 cKO mouse model to investigate ADAM10\\u0026rsquo;s role in APP shedding.\\u003c/p\\u003e \\u003cp\\u003eAβ peptides are constitutively secreted by nearly all neuronal cells and non-neuronal cells expressing APP. Among these, Aβ40, Aβ42, and Aβ43 represent the primary neurotoxic species, with Aβ42 and Aβ43 exhibiting enhanced fibrillation propensity. Aβ peptides are the primary component of senile plaques. Current evidence suggests that excessive production of Aβ peptides or impaired clearance pathways leading to amyloid-beta deposition is a critical mechanism underlying AD pathogenesis [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. Our data demonstrate decreased sAPPα and C83 levels, along with increased the levels of sAPPβ, C99, total Aβ, Aβ40 and Aβ42/43 in the cortex and hippocampus of the \\u003cem\\u003eADAM10\\u003c/em\\u003e cKO mice. The immunohistochemistry results showed an increase in soluble Aβ levels within neurons in the cerebral cortex and hippocampus of ADAM10 cKO mice. This may be attributed to reduced α-secretase activity of ADAM10, coupled with upregulated β-secretase activity, which shifts APP processing toward β-cleavage. However, our experimental data cannot yet rule out disrupted APP processing or clearance, or altered APP subcellular localization. These findings indirectly suggest that there may be a competitive relationship between ADAM10 α-secretase and β-secretase for APP substrate processing in mice.\\u003c/p\\u003e \\u003cp\\u003eSynapses play a role in the transmission and transformation of information in neural circuits. To investigate the impact of ADAM10 deletion on synaptic function, we analyzed synaptophysin and PSD-95 expression in the cerebral cortex and hippocampus of the ADAM10 cKO mice, assessed synaptic ultrastructure in the hippocampus, and measured LTP in hippocampal neurons.\\u003c/p\\u003e \\u003cp\\u003eSynaptophysin,a 38 kDa transmembrane glycoprotein with four transmembrane domains, serves as a presynaptic vesicle marker and is involved in the formation and exocytosis of synaptic vesicles [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. It regulates neurotransmitter release, a process vital for interneuronal communication. Hubert Rehm et al. demonstrated that synaptophysin acts as the primary calcium-binding protein of synaptic vesicles and plays an important role in the release of neurotransmitters [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. Additionally, synaptophysin interacts with synaptobrevin to form a complex that facilitates functional SNARE assembly, thereby regulating synaptic efficacy [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. As a presynaptic terminal marker, synaptophysin is widely used in studies of neurodegenerative diseases (e.g., Parkinson\\u0026rsquo;s disease, AD, and dementia) and transgenic models. Its investigation provides insights into synaptic dysfunction and potential therapeutic strategies. Notably, hippocampal synaptophysin levels are significantly reduced in AD patients, correlating with severe cognitive deficits [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e]. Similarly, Oakley et al. reported age-dependent synaptophysin decline and memory impairment in transgenic mice overexpressing familial AD-linked APP/PS1 mutations [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. The study by Cao Y et al. demonstrated that synaptophysin levels are reduced in both AD patients and transgenic mice carrying the amyloid precursor mutation (AβPP) 670/671 (AβPPSWE). Furthermore, bilateral hippocampal injection of Aβ42 in mice resulted in decreased synaptophysin expression at both protein and mRNA levels, accompanied by learning and spatial memory deficits [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. Our findings in the ADAM10 cKO mouse model suggest that reduced synaptophysin expression in the cortex and hippocampus of these mice impairs interneuronal communication, thereby contributing to their learning and memory dysfunction.\\u003c/p\\u003e \\u003cp\\u003eThe primary role of postsynaptic element is to receive neurotransmitters released from presynaptic terminals. A uniform, dense layer of material called the postsynaptic density (PSD) is observed on the cytoplasmic surface of the postsynaptic membrane. And PSD-95 is a key scaffolding protein located in the PSD. PSD-95 consists of of three PDZ domains, Src homology 3 (SH3) domain, and a non-catalytic guanylate kinase domain [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. Through it\\u0026rsquo;s PDZ domains, PSD-95 binds to glutamate receptors, anchoring glutamate them to the PSD organizing downstream signaling molecules around these receptors [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. Thus, PSD-95 is critical for synaptic signal transduction and functional integrity. At present, studies on PSD-95 expression in AD reveal conflicting results. Gylys et al.,reported decreased PSD-95 levels in the cerebral cortex of AD patients [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e] while Love et al. similarly observed reduced PSD-95 in the cortical regions [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. In contrast, Leuba et al. found elevated PSD-95 expression in the entorhinal cortex of AD patients [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e], and Gong et al. reported increased PSD-95 in synaptosomes from AD brains [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. In transgenic animal models, PSD-95 expressionvaries regionally. SAMP10 mice (a brain aging model) exhibit reduced cortical PSD-95 but unchanged levels in the brainstem [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. Similarly, in 5XFAD and JNPL3 AD mouse models, PSD-95 decreases in the apical dendrites of hippocampal CA1 neurons in 6-month-old 5XFAD and 14-month-old JNPL3 mice, yet increases in neuronal soma [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. These dynamics suggest PSD-95 expression changes dynamically during disease progression. Reduced PSD-95 may reflect synaptic vulnerability to Aβ peptide toxicity [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e], while compensatory upregulation could indicate de novo synthesis to mitigate synaptic damage [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]. In our study on the ADAM10 cKO mouse model, we found no significant difference in the expression level of PSD-95 in the cortex and hippocampal CA3 region, while the expression of PSD-95 in the hippocampal CA1 region was decreased. Additionally, levels of Aβ40 and Aβ42 increased in the hippocampus in the hippocampus, along with abnormal synaptic structures. In the hippocampus of ADAM10 cKO mice, the accumulation of Aβ increased. Aβ can strongly bind to the neuronal receptor PirB, triggering intercellular signaling processes that erode neuronal synapses, leading to synaptic contact impairment or even loss. Ultimately, this results in reduced PSD-95 levels in the hippocampal CA1 and impaired learning and memory abilities in the mouse model.\\u003c/p\\u003e \\u003cp\\u003eSynaptic plasticity refers to property that that allows for more lasting changes in synaptic efficacy, encompassing structural plasticity and functional plasticity, which are interrelated and mutually influence. The plasticity of synaptic structure is manifested as changes in synaptic morphology, including the curvature of the synaptic interface, the thickness of the synaptic dense zone, the width of the synaptic cleft, the synaptic number, and the density of synaptic area. The plasticity of synaptic function is manifested by the enhancement or weakening of synaptic information transmission capacity, such as LTP. Studies of synaptic ultrastructure in the hippocampal CA1 region of a mouse model of neurofibromatosis type I (a neurological dysfunction disorder) have shown that synaptic interface curvature is lower than in normal mice, underscoring the importance of synaptic interface curvature in synaptic plasticity, learning and memory function [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e]. Cerebral vascular hypoperfusion experiments in mice revealed the spatial memory impairment and altered synaptic ultrastructure in the hippocampal CA1 region after 30 days [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e]. Studies of the presenilin (PS) conditional knockout mouse model demonstrated impaired synaptic plasticity, learning and memory, along with inhibited LTP induction in multiple brain regions [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e]. Therefore, our study investigating synaptic ultrastructure and LTP induction in the hippocampal CA1 region of the ADAM10 cKO mice may provide further insights into the mechanisms underling learning behavior in these mice.Our fingdings demonstrate ultrastructural synaptic abnormalities and impaired LTP in hippocampal neurons of ADAM10 cKO mice. The hippocampal CA1 region as the terminal hub of excitatory synaptic circuits, plays a critical role in memory-related synaptic networks. Dysfunction in this region contributes to memory impairment and neurological disorders, which may explain why the hippocampus CA1 region is particularly vulnerable to pathological factors associated with dementia. Regarding the mechanistic basis of these observations, ADAM10 may interact with synaptic-associated protein 97 (SAP97) to regulate synaptic function and formation. Genetic deletion of ADAM10 resulted in synaptic dysfunction, manifested by reduced expression levels of synaptophysin and PSD-95, as well as ultrastructural synaptic abnormalities. These defects likely represent key contributors to the spatial learning and memory deficits observed in the ADAM10 cKO mice.\\u003c/p\\u003e \\u003cp\\u003eAstrocytic proliferation is a pathological hallmark of structural lesions in the central nervous system (CNS) [\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e]. When the CNS suffers severe damage or disease, an inflammatory response may occur, accompanied by the process of astrocytic proliferation. This process is primarily characterized by upregulated GFAP protein expression, hypertrophy of glial cells, and an increase in their numbers [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e]. Astrocytes can accumulate large amounts of Aβ peptides derived from neuronal cells and degrade them in response to localized neurodegeneration [\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e][\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e]. Studies on transgenic mice overexpressing Aβ peptides in neurons have demonstrated the neurotoxicity of Aβ peptides, showing extensive neuronal degeneration alongside increased glial cell proliferation, suggesting that neuronal loss is caused by apoptosis [\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e]. Our study revealed that significant upregulation of GFAP expression was observed in both the cerebral cortex and hippocampus, indicating astrocyte activation. Despite the absence of amyloid plaque deposition, neurons exhibited increased accumulation of soluble Aβ species. Notably elevated levels of neurotoxic Aβ40 and Aβ42 were detected in these regions, with evident oligomerization. This Aβ accumulation triggered neuroinflammatory responses and reactive astrogliosis, ultimately leading to neuronal damage and degeneration. Hippocampal CA1 neurons showed reduced PSD thickness. LTP magnitude was significantly attenuated following high-frequency stimulation. Morris water maze tests revealed impaired spatial learning and memory in our ADAM10 cKO mice, and quantitative stereology demonstrated reduced granule cell numbers in the hippocampal formation [\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e]. These findings suggest the Aβ-mediated neuroinflammatory cascade in ADAM10 cKO mice manifests through: (i) astrocyte activation (\\u0026uarr;GFAP), (ii) synaptic ultrastructural defects (\\u0026darr;PSD thickness), (iii) functional plasticity impairment (\\u0026darr;LTP magnitude), and (iv) ultimately, cognitive decline. Certainly, in future studies, we will need to further clarify the direct relationship between Aβ, neuroinflammation, and synaptic damage through experiments examining Aβ synaptic localization and inflammation modulation-mediated restoration of synaptic marker expression.\\u003c/p\\u003e \\u003c/li\\u003e \\u003c/ul\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn summary, we provided evidence in vivo that ADAM10 gene deletion has remarkable effects on APP processing in the adult mouse brain. The ADAM10 gene is essential for the formation and maintenance of synaptic plasticity. It\\u0026rsquo;s deletion results in synaptic abnormalities,which severely affect information transmission between neurons, leading to impaired learning and memory in adult mice. These results will help us deepen our understanding of ADAM10\\u0026rsquo;s function and provide a theoretical basis for the prevention and treatment of AD.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003ctable border=\\\"0\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"568\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eADAM10\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eA disintegrin and metalloprotease10\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAPP\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAmyloid precursor protein\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003ecKO\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003econditional knock out\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eDG\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003edentate gyrus\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003emin\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eminute\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eELISA\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eenzyme linked immunosorbent assay\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003es\\u003c/strong\\u003e\\u003cstrong\\u003eAPP\\u0026beta;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003esoluble APP\\u0026beta;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003es\\u003c/strong\\u003e\\u003cstrong\\u003eAPP\\u0026alpha;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003esoluble\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;APP\\u0026alpha;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eC99\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eC-terminal fragment 99\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eC83\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eC-terminal fragment 83\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAICD\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAPP intracellular domains\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eA\\u0026beta;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAmyloid-beta\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eLTP\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003elong-term potentiation\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003ePSD\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003epostsynaptic dense\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAD\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAlzheimer\\u0026apos;s Disease\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eEPSP\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eexcitatory postsynaptic potential\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eTEM\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003etransmission electron microscopy\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eMOD\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003emean optical density\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eHFS\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003etotal FL-APP\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003etotal sAPP\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003emAPP\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eimAPP\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003ehigh-frequency stimulus\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003etotal full-length APP\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003etotal soluble APP\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003emature APP\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eimmature APP\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 31.8662%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eGFAP\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 68.1338%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eg\\u003c/strong\\u003e\\u003cstrong\\u003elial fibrillary acidic protein\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eConsent to\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ePublish declaration\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll the authors agree for publication\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eif our article is accepted for publication.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDate Availability\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eD\\u003c/strong\\u003e\\u003cstrong\\u003eeclaration\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent to Participate declaration: not applicable\\u003c/strong\\u003e\\u003cstrong\\u003e.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eD\\u003c/strong\\u003e\\u003cstrong\\u003eeclaration\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll mice were raised by the Laboratory Animal Center, Fujian Medical University following the general guideline of AAALAC handbook and institutional regulations.\\u0026nbsp;In accordance with ARRIVE guidelines, the method we used for euthanasia of mice was cervical dislocation.\\u0026nbsp;The experimental procedures for the use and care of animals were approved by the Ethics Committee of Fujian Medical University (Permit Number: FMU Ethical Inspection N.2009-32) , China.\\u0026nbsp;All experiments were performed in accordance with relevant guidelines and regulations.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting Interests\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eD\\u003c/strong\\u003e\\u003cstrong\\u003eeclaration\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eI declare that the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor Contributions\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eD\\u003c/strong\\u003e\\u003cstrong\\u003eeclaration\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eQ.W. wrote the main manuscript text, analyzed and processed the data and graphs, prepared figures 1,4,5. S.L.prepared figures 2-3. Y.L. prepared figures 6-10, P.L. prepared figures 10, J.H. participated in the processing of the images and data. Corresponding authors C.Z. and Z.H. guided the experiments and the writing of the article. All authors reviewed the manuscript. Q.W., S.L. and Y.L. contributed equally to this work.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp; Acknowledgement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe thank Professor Yuan Zhuang and Professor Xiaohui Wu of the Institute of Developmental Biology and Molecular Medicine of Fudan University for providing ADAM10\\u003csup\\u003eloxP/loxP\\u003c/sup\\u003e transgenic mice for this experiment, the teachers of the Electron Microscope Room of Fujian Medical University for their guidance and assistance, and Professor Fenghua Lan and Dr. Yuqi Zeng of Fujian Medical University for their guidance and help in the experiments.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by\\u0026nbsp;Natural Science Foundation of Fujian\\u0026nbsp;Province of China Grant\\u0026nbsp;2010j01171 and\\u0026nbsp;Key project of Experimental animal research of Fujian\\u0026nbsp;Province of China Grant\\u0026nbsp;2014Y0078.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eMohamed Asik R, Suganthy N, Aarifa MA, Kumar A, Szigeti K, Mathe D, Guly\\u0026aacute;s B, Archunan G, Padmanabhan P. Alzheimer's Disease: A Molecular View of β-Amyloid Induced Morbific Events. Biomedicines. 2021;9(9):1126.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhang YW, Thompson R, Zhang H, Xu H. APP processing in Alzheimer's disease. Mol Brain. 2011;4:3.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSastre M, Walter J, Gentleman SM. Interactions between APP secretases and inflammatory mediators. J Neuroinflammation. 2008;5:25.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLichtenthaler SF. α-secretase in Alzheimer's disease: molecular identity, regulation and therapeutic potential. J Neurochem. 2011;116(1):10\\u0026ndash;21.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhang C, Saunders AJ. Therapeutic targeting of the alpha-secretase pathway to treat Alzheimer's disease. 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J Neurosci. 2010;30(14):4833\\u0026ndash;44.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eColombo A, Wang H, Kuhn PH, Page R, Kremmer E, Dempsey PJ, Crawford HC, Lichtenthaler SF. Constitutive α- and β-secretase cleavages of the amyloid precursor protein are partially coupled in neurons, but not in frequently used cell lines. Neurobiol Dis. 2013;49:137\\u0026ndash;47.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eProx J, Bernreuther C, Altmeppen H, Grendel J, Glatzel M, D'Hooge R, et al. Postnatal disruption of the disintegrin/metalloproteinase ADAM10 in brain causes epileptic seizures, learning deficits, altered spine morphology, and defective synaptic functions[J]. Neurosci. Aug 2013;7(32):12915\\u0026ndash;28.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePeron R, Vatanabe IP, Manzine PR, Camins A, Cominetti MR. Alpha-Secretase ADAM10 Regulation: Insights into Alzheimer's Disease Treatment. 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Acta Neuropathol. 2010;119(1):7\\u0026ndash;35.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGuillam\\u0026oacute;n-Vivancos T, G\\u0026oacute;mez-Pinedo U, Mat\\u0026iacute;as-Guiu J. Astrocytes in neurodegenerative diseases (I): function and molecular description. Neurologia. 2015;30(2):119\\u0026ndash;29.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eNagele RG, Wegiel J, Venkataraman V, Imaki H, Wang KC, Wegiel J. Contribution of glial cells to the development of amyloid plaques in Alzheimer's disease. Neurobiol Aging. 2004 May-Jun;25(5):663\\u0026thinsp;\\u0026ndash;\\u0026thinsp;74.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYang Z, Wang KK. Glial fibrillary acidic protein: from intermediate filament assembly and gliosis to neurobiomarker. Trends Neurosci. 2015;38(6):364\\u0026ndash;74.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLaFerla FM, Tinkle BT, Bieberich CJ, Haudenschild CC, Jay G. The Alzheimer's A beta peptide induces neurodegeneration and apoptotic cell death in transgenic mice. Nat Genet. 1995;9(1):21\\u0026ndash;30.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhuang J, Wei Q, Lin Z, Zhou C. Effects of ADAM10 deletion on Notch-1 signaling pathway and neuronal maintenance in adult mouse brain. Gene. 2015;555(2):150\\u0026ndash;8.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"deintegrin and metalloproteinase 10 (ADAM10), amyloid precursor protein (APP), synapses, long-term potentiation (LTP)\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8193475/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8193475/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eA disintegrin and metalloproteinase 10 (ADAM10) is a member of the ADAMs (a deintegrin and metalloproteinases) family, which hydrolyzes various cellular receptors and signaling molecules (such as APP) to regulate the development of body organs and tissues. As ADAM10 is a susceptibility gene for Alzheimer's disease (AD), we generated adult neural cell-specific \\u003cem\\u003eADAM10\\u003c/em\\u003e conditional knockout (ADAM10 cKO) mice to study its effects on APP shedding, synaptic integrity, and cognitive function. Our results revealed that ADAM10 deletion significantly increased levels of sAPPβ, C99, total Aβ peptides, while decreasing levels of sAPPα and C83 in the hippocampus and cortex. These changes trigger neuroinflammation, as evidenced by elevated GFAP expression. Furthermore, synaptophysin expression was reduced in the hippocampus and cortex, with postsynaptic density protein-95 (PSD-95) showing decreased expression in the hippocampal CA1 region. Synaptic ultrastructure exhibited abnormalities, and long-term potentiation (LTP) induction was impaired, ultimately leading to learning and memory deficits. These findings advance our understanding of ADAM10's biological functions and provide mechanistic insights for AD prevention and therapeutic strategies.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Effects of ADAM10 deletion on APP shedding, synapse and synaptic plasticity in adult mice\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-12-16 14:12:07\",\"doi\":\"10.21203/rs.3.rs-8193475/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"f29907bb-3a10-4e9a-a70b-53c8fdb8153b\",\"owner\":[],\"postedDate\":\"December 16th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-03-27T12:10:13+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-12-16 14:12:07\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8193475\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8193475\",\"identity\":\"rs-8193475\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}