Autophagy and neuroinflammation modulation by nano-graphene oxide in PSEN1 Alzheimer’s brain organoids

Autophagy and neuroinflammation modulation by nano-graphene oxide in PSEN1 Alzheimer’s brain organoids | 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 Research Article Autophagy and neuroinflammation modulation by nano-graphene oxide in PSEN1 Alzheimer’s brain organoids Nam Gyo Kim, Jaeyong Chun, Hyeyeon Park, Hyunyoung Kim, Soon Won Choi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8383146/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract Background Alzheimer’s disease (AD) is one of the best-known neurodegenerative diseases, and substantial progress has been made in the field of neuroscience and AD. However, there has been no major improvement in AD treatment. Methods Cerebral organoids were generated using induced pluripotent stem cells (iPSCs) from both healthy individual and AD patients. Organoids were cultured for 12 weeks and then nano-graphene oxide (NGO) was treated for another 2 weeks. Organoids were then sampled according to different sampling methods for various analysis. Results AD patient-derived iPSCs were employed to generate cerebral brain organoids and were cultured until they expressed AD phenotypes, accumulation of amyloid-beta (Aβ) plaquesand hyperphosphorylated tau (pTau)-derived neurofibrillary tangles (NFTs). We investigated whether NGO treatment could decrease the expression of the major hallmarks of AD. Our study illustrates that accumulated Aβ plaques were diminished in the NGO-treated AD organoids. NGO activates the autophagy pathway, targeting AMPK activation. The changes in the levels of AMP and ADP in NGO treated group suggest that NGO affects cellular energy metabolism, thus activating AMPK autophagic pathway. Also, the astrocytes and microglial cell presence in our model allowed examining further into inflammatory effect of NGO. The decrease in proinflammatory cytokine levels in NGO treated group led to decrease in IFITM3 expression and going further into diminishing γ-secretase activity. Conclusions Overall, the development of AD brain organoids successfully mimickedAD phenotype expression;thus, they could be used as a screening platform for novel AD treatment assessments. Alzheimer's Disease Induced Pluripotent Stem Cells Brain Organoid Nano Graphene Oxide Autophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 BACKGROUND Alzheimer’s disease (AD) is a neurodegenerative disease affecting more than 50 million individuals worldwide, and its research is a rapidly growing field that strives to identify the underlying causes of AD phenotypes, as well as to develop effective drugs or treatments. In terms of familial AD (FAD), patients tend to develop AD symptoms at much younger ages than sporadic AD (SAD) patients [ 1 ]. Autosomal dominant genetic mutations such as amyloid precursor protein (APP) or presenilin-1 (PSEN1) mutations are known to cause the most severe forms of AD [ 2 ]. Researchers have been utilizing a variety of techniques, including genetic studies, brain imaging, and animal models, to learn more about the changes that occur inside the brain as AD progresses [ 3 ]. The major hallmark of AD is now known as hyperphosphorylated tau (pTau)-derived neurofibrillary tangles (NFTs) and the neuronal accumulation and consequent toxicity of amyloid-beta (Aβ) plaques [ 4 ]. These establishments have aided in clinical trials of new ongoing treatments; however, despite extensive progress in the neuroscience of AD, no significant improvements were made in the areas of AD treatments [ 5 ]. In the past couple of decades, there have been multiple attempts at manufacturing an in vitro experimental platform to mimic the structural and functional architectures of the human brain [ 6 ]. However, creating a reproducible research platform which could potentially incorporate fully or even partially mimicking the human brain has been very challenging [ 7 ]. In order to overcome such challenges, the use of stem cells and 3D in vitro modelling systems have been brought up by multiple research groups. Brain organoids are 3D cultures of brain cells derived from induced pluripotent stem cells (iPSCs) [ 8 ]. These organoids offer a number of advantages for studying the brain and neurological diseases [ 9 ]. For instance, organoids provide a way to study brain development and the underlying mechanisms of certain neurodegenerative diseases, such as Parkinson’s and Alzheimer’s diseases, in a controlled and realistic environment [ 10 ]. Moreover, brain organoids provide special platforms where one could elaborate on utilizing viral infections, such as Zika and SARS-CoV-2 infection, and examine different phenotypes caused by specific viral infection [ 8 , 11 ]. Even though mouse models are a frequently used research model in neurodegenerative disease research, given the major differences between different species, phenotypes displayed in the human AD brain are not fully represented in the mouse brain AD model [ 12 ]. Graphene, a hexagonal lattice of carbon atoms, displays varied properties influenced by different size and functional groups [ 13 ]. Graphene oxide (GO), a derivative modified with oxygen-containing groups like carboxyl and hydroxyl, and nano-graphene oxide (NGO), with dimensions below 100 nm and depth of only couple of sheets, exhibit unique characteristics over conventional GO; such as quantum confinement and edge effects [ 14 ]. Due to their compatibility with biological systems and ability to modulate immune responses, NGOs have gained traction in biomedical applications [ 15 ]. Despite efforts to incorporate GO into reinforcing biopolymers, still remains a lack of understanding regarding the in vivo effects of NGOs, particularly in the context of excretion [ 16 ]. As the therapeutic use of nanoparticles strongly depends on the size of the particles, we have manufactured GO with average lateral size of 10nm and named as nano-graphene oxide (NGO) [ 14 ]. The purpose of this study was to assess the effectiveness of NGO on AD cerebral brain organoids produced from patient-derived iPSCs. We hypothesized that NGO could potentially reduce AD phenotypes by activating autophagic mechanisms and diminishing neuroinflammation. Overall, the results suggest that this AD brain organoid model can serve as a useful tool to test treatments for AD patients. METHODS iPSC Culture We obtained five human-iPSC cell lines for this study. One wildtype iPSCs; CMC-hiPSC-003 (WT-CMC3), and two AD patient-derived iPSCs; UCSD241i-APP2-3 (241AD) and SCD234i-SAD2-3 (234AD), were provided by National Stem Cell Bank of Korea (Korea National Institute of Health, South Korea), and for the PSEN1-mutated patient-derived iPSC line (PSEN1 AD), the CS40iFAD-nxx (RRID:CVCL_YX94) cell line was purchased from Cedar-Sinai Medical Center iPSC Core Facility. Also, another wildtype iPSC; WT-Maru, was provided by Maru Therapeutics (Maru Therapeutics, Seoul, South Korea). Both WT-iPSCs and AD-iPSCs were used to generate wild-type and AD cerebral brain organoids, respectively. WT-CMC3 iPSCs were cultured on vitronectin (Gibco, Massachusetts, USA)-coated plates and cultured using Essential 8™ Medium (Gibco, Massachusetts, USA), WT-Maru iPSCs were cultured on GFR-Matrigel (Corning, Massachusetts, USA)-coated plates using STEMFLEX™ Medium (Gibco, Massachusetts, USA), PSEN1 iPSCs were cultured on GFR-Matrigel (Corning, Massachusetts, USA)-coated plates and cultured using mTeSR TM -Plus medium (STEMCELL Technologies, Canada), 241AD iPSCs were cultured on GFR-Matrigel (Corning, Massachusetts, USA)-coated plates and cultured using mTeSR TM -Plus medium (STEMCELL Technologies, Canada) and 234AD iPSCs were cultured vitronectin (Gibco, Massachusetts, USA)-coated plates and cultured using E8-mTeSR™ medium (STEMCELL Technologies, Canada) as instructed by the manufacturers. All of the cell lines were passaged using ReLeSR™ (STEMCELL Technologies, Canada) when confluent. Brain Organoid Generation Generation of brain organoids was performed following the protocol previously described by Lancaster et al. [ 17 ]. In brief, iPSCs were dissociated into single cells using TryPLE™ express enzyme (Thermo Fisher Scientific, Massachusetts, USA). Dissociated iPSCs were then seeded onto ultralow binding 96-well plates (Corning, Massachusetts, USA), with 9,000 cells in each well with mTeSR TM -Plus medium, 4 ng/ml basic fibroblast growth factor (Sigma‒Aldrich, Missouri, USA) and 50 µM Rho-associated protein kinase inhibitor. Then, the medium was replaced daily with fresh medium when EBs were formed. Then, as the diameter of EBs reached 500 µm, they were transferred to low-adhesion 24-well plates (Corning, Massachusetts, USA) with neural induction medium consisting of DMEM-F12 (Gibco, Massachusetts, USA) with 1x N-2 Supplement (Gibco, Massachusetts, USA), 1x GlutaMAX (Gibco, Massachusetts, USA), 1x MEM-NEAA (Gibco, Massachusetts, USA) and 1 µg/ml heparin solution. After 6 days, neuroectodermal tissues were embedded in Matrigel droplets (Corning, Massachusetts, USA). These droplets were incubated at 37°C for 20 minutes to solidify and cultured in cerebral organoid differentiation medium without vitamin A (CODM-wo) consisting of a 1:1 ratio of DMEM-F12 (Gibco, Massachusetts, USA) and Neurobasal Medium (Gibco, Massachusetts, USA) with 1x N-2 Supplement (Gibco, Massachusetts, USA), 1x B-27 Supplement without Vitamin A (Gibco, Massachusetts, USA), 1x 2-mercaptoethanol (Sigma-Aldrich, USA), 1x insulin (Sigma-Aldrich, USA), 1x GlutaMAX (Gibco, Massachusetts, USA) and 1x MEM-NEAA (Gibco, Massachusetts, USA) for 4 days. Then, the medium was replaced with CODM composed of a 1:1 ratio of DMEM-F12 (Gibco, Massachusetts, USA) and Neurobasal Medium (Gibco, Massachusetts, USA) with 1x N-2 Supplement (Gibco, Massachusetts, USA), 1x B-27 Supplement (Gibco, Massachusetts, USA), 1x 2-mercaptoethanol (Sigma-Aldrich, USA), 1x insulin (Sigma-Aldrich, USA), 1x GlutaMAX (Gibco, Massachusetts, USA) and 1x MEM-NEAA (Gibco, Massachusetts, USA). NGO and NGO-biotin Production and Characterization NGO was produced and provided by INBCT Co., LTD (Seoul, Korea). NGO was synthesized from graphite via Taylor-Couette flow and stabilized solid state of NGO powder was suspended in deionized water. NGO was then loaded onto a 400-mesh carbon coated cooper grid for morphology analysis using Cs-corrected HRTEM (JEM-ARM200F, Cold FEG, JEOL Ltd, Japan). The size distribution of NGO was analyzed using a CPS disc centrifuge (CPS Instruments, USA). Next, NGO was then loaded onto sapphire wafers and then scanned by atomic force microscopy in noncontact mode (scanned area 25 µm 2 , XE-100, Park Systems, Korea). NGO was also provided by INBCT Co., LTD (Seoul, Korea). The method of producing biotin tagged NGO followed similar protocol as producing NGO, with an extra modification and sterilization using high resistance nonspecific probe displacement technique. NGO and NGO-biotin Treatment On Day 84, both WT and AD brain organoids were exposed to NGO or NGO-biotin at a concentration of 10 µg/ml. Organoids were treated for 2 weeks with the CODM + NGO medium or CODM + NGO-biotin medium, changed every other day. MTT Assay iNSCs were seeded at 5 x 10 4 cells/well in a 24-well plate in iNSC maintenance medium. After 24 h, cells were treated with A-769662 at different concentrations for 48 h. Then, 500 µl of MTT solution at 500 µg/ml concentration was added to each well and incubated for 4 h at 37°C. Then, 500 µl of DMSO was added to each well to dissolve the formazan crystals. The absorbance value at 570 nm was obtained using an Infinite200 PRO microplate reader (Tecan, Switzerland). RNA Extraction and Reverse Transcriptase PCR Three cerebral brain organoids in each experimental group were lysed in 1 ml of TRIzol (Invitrogen, Massachusetts, USA) for RNA extraction according to the manufacturer’s instructions. cDNA synthesis was performed using Superscript-III First-Strand KIT (Invitrogen, Massachusetts, USA) for RT‒PCR analysis. RT‒PCR was performed with SYBR Green PCR Mix (Applied Biosystems, Massachusetts, USA) using a 7500 Real-Time PCR system (Thermo Fisher Scientific, Massachusetts, USA) carried out with initial denaturation conditions at 50°C for 2 min and at 95°C for 10 min followed by 95°C for 15 s and 60°C for 1 min for at least 60 cycles. All readings were normalized to the level of GAPDH, the housekeeping gene. The list of primers used, and their sequences are listed in Supplementary Table 1. Western Blot Analysis Western Blot Analysis Three cerebral brain organoids in each experimental group were lysed with 300 µl of Pro-Prep protein lysis buffer (Intron Biotechnology, South Korea) and Sonifier450 (Branson, Connecticut, USA). The concentration of protein samples was measured using a Bio-Rad DC protein assay kit (Bio-Rad Laboratories, California, USA), alongside bovine serum albumin (BSA) as the standard. Each sample was separated using 6%, 10% and 15% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and transferred onto a nitrocellulose membrane. The membrane was then blocked with 3% BSA and incubated overnight with a single primary antibody at 4°C. After another 1 h incubation with the appropriate secondary antibody, the proteins were detected with enhanced chemiluminescence (ECL) reagent (GE Healthcare Life Science, UK) and then viewed using ChemiDocTM MP (Bio-RAD, California, USA) Histology and Immunofluorescence Organoids were fixed in 4% paraformaldehyde for 1h at RT. Each organoid was washed with PBS and incubated in 30% sucrose solution for at least 24h at 4°C. The sample was then embedded in sucrose/gelatin solution and rapidly frozen using liquid nitrogen. Samples were then sectioned into thin slices and used for immunostaining. Sectioned samples were washed and then placed under permeabilization and blocking solution. Each sample was incubated with specific primary antibodies overnight at 4°C. After another 1h incubation with matching secondary antibodies, the samples were then stained with DAPI and mounted using DAKO fluorescence mounting medium (Agilent Pathology Solutions, California, USA). ELISA The protein levels of Aβ40 and Aβ42 in RIPA buffer were measured using human Aβ40 ELISA Kit (Thermo Fisher Scientific, Massachusetts, USA) and Aβ42 ELISA Kit (Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturer’s instructions and quantified using BCA analysis. RIPA lysates of 3 cerebral brain organoids were used to create a single reading of Aβ40 and Aβ42 and each group was measured in triplicate. ADP/ATP Ratio Assay The ADP/ATP ratio assay was performed according to the manufacturer’s instructions. In short, 3 cerebral brain organoids per group were dissociated using Accutase and seeded onto 96-well plates at 10 4 cells per well. ATP reagent mix was added to each well and incubated for 1 min at RT. Then, ATP luminescence was recorded, RLU A . After another 10 min incubation at RT, ADP luminescence was recorded, RLU B . Immediately after the ADP luminescence was recorded, ADP reagent mix was added and incubated for 1 min at RT, and then, luminescence was recorded, RLU C . All experiments were performed in triplicate. AMP Colorimetric Assay The AMP colorimetric assay was performed according to the manufacturer’s instructions. In short, 3 cerebral brain organoids per group were dissociated using AMP assay buffer. After centrifugation at 10,000 g at 4°C for 10 min, the supernatant was collected, and 20 µl was placed onto a 96-well plate with 30 µl of AMP assay buffer. Then, 50 µl of reaction mix was added to each well and incubated for 60 min at 37°C. After incubation, absorbance was recorded at 570 nm. All experiments were performed in triplicate. QUANTSeq mRNA-Sequencing Analysis mRNA sequencing analysis was performed on 2 samples per group: WT Cont, WT NGO, PSEN1 Cont and PSEN1 NGO. Each RNA sample was created using 3 organoids undergoing total RNA isolation using 1ml of TRIzol (Invitrogen, Massachusetts, USA) according to the manufacturer’s instructions. All of the RNA samples’ quality control was done using Bioanalyzer 2100 system using ND-2000 spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA). Each sample underwent cDNA library construction and sequencing using a QuantSeq 3′ mRNA-Seq Library Prep Kit (Lexogen Inc., Austraia), following the manufacturer’s protocol. High-throughput sequencing was conducted as single-end 75, generating approximately 10 million reads per sample, utilizing a NextSeq 500 (Illumina Inc., California, USA). For further analysis, ExDEGA (eBiogen, South Korea) gene analysis tool was used to determine differentially expressed genes with fold change equal or higher than 2 and p-value of equal or less than 0.05. The clustering heatmaps and gene ontology data were created using SRplot. Statistical Analysis Data analyses in all related experiments were carried out with Prism 9 software (GraphPad Software, California, USA), and statistical analyses were performed depending on the number of groups, distribution, variance, and normality. Two-tailed Student’s t test analysis and one-way ANOVA were employed for parametric datasets, and the level of significance is shown as *** P < 0.001, ** P < 0.01, or * P < 0.05 or ns for not significant. The number of biological replications performed in different experiments is shown in each figure legend. Furthermore, all samples were chosen randomly for analysis. RESULTS Generation and characterization of WT and PSEN1 patient-derived cerebral brain organoids In this study, two iPSC lines were employed to simulate AD. CMC-hiPSC-003 cells were utilized as the WT-CMC3 control group, while PSEN1 iPSCs were used as the experimental group (Fig. 1 A). These cell lines were used to generate cerebral brain organoids using a protocol that was previously established with slight modifications [ 17 ]. Briefly, approximately 9000 iPSCs were seeded onto a single ultralow-attachment 96-well plate for 6 days to form an embryoid body. On Day 6, EBs were collected and underwent neural induction for 4 days. On Day 11, each organoid was placed inside a Matrigel droplet placed in CODM medium without vitamin A for neuroepithelial bud expansion and then transferred to CODM medium with vitamin A for further expansion. Both WT-CMC3 and PSEN1 AD organoids were cultured for up to 12 to 14 weeks of maturation and then sampled at different time points for further analysis (Fig. 1 B). The size of the brain organoids from both WT-CMC3 and PSEN1 mice showed fairly even growth. The PSEN1 AD brain organoids were slightly smaller than the WT-CMC3 organoids on Day 56; however, both groups showed similar sizes on Day 84 (Supple Fig. 1 B). The average area of the organoids on Day 42 was determined, and sections of the WT-CMC3 and PSEN1 AD brain organoids both showed clear expression of the cell proliferation marker KI-67, neuronal progenitor marker SOX2, and neuronal markers TUJ1 and MAP2. These markers were also expressed on Day 84 in both the WT-CMC3 and PSEN1 AD brain organoids (Fig. 1 C). These immunostaining images illustrate that brain organoids generated from PSEN1-mutated patient-derived iPSCs exhibited similar levels of brain development compared to WT-CMC3 brain organoids. The results of quantifying specific markers through ImageJ software also showed that the development and maturity of the PSEN1 AD brain organoids were comparable to those of the WT brain organoids (Fig. 1 D). Collectively, both the WT-CMC3 and PSEN1 AD brain organoids effectively mimicked neurodevelopment in organoid cultures. PSEN1 AD brain organoids were generated using iPSCs derived from an AD patient with the PSEN1 A246E mutation. This mutation is known to have altered catalytic site conformation on γ-secretase, which results in an impaired 4th cleavage site. This alteration affects the cleavage site of APP, which then leads to an increase in the ratio of Aβ42/Aβ40 (Fig. 2 A). With this genetic alteration, PSEN1 AD brain organoids are expected to show an increase in AD phenotypes. As shown in Fig. 2 B, Tau immunostaining was observed in both the WT-CMC3 and PSEN1 AD brain organoids at all time points. In the case of the AD phenotypic markers pTau and Aβ, neither marker was expressed in the WT-CMC3 brain organoids at either Day 42 or Day 84. In the PSEN1 AD brain organoid samples, pTau and Aβ were expressed (Fig. 2 B). The quantified expression of AD phenotypic markers illustrate that Aβ tend to increase at a faster rate when cultured for a longer period of time. On the other hand, the growth of pTau expression tends to occur but at much slower rate than Aβ (Fig. 2 C). Nano-graphene oxide characterization In order to categorize NGO used in this study as nanoparticles, particle size and the distribution were measured. Atomic force microscopy (AFM) was used to measure the thickness of NGO. AFM measurement showed that the thickness of NGO is around 0.72nm and 0.77nm (Fig. 3 A). Since the thickness of a single sheet layer of graphene oxide is to be below 1.2nm, AFM shows that NGO is composed of single layer NGOs [ 16 ]. Transmission electron microscopy (TEM) method was then used to measure the size of the NGO. TEM image illustrates that most of the NGO particles are smaller than 20nm scale bar used in the image (Fig. 3 B). We also measured about 2.8x10 12 particles in total and created a lateral size distribution graph which states the average size of each NGO particle is around 10nm and that 99% of the particles are distributed within the range of 5nm to 33.7nm (Supple Fig. 2 A). Fourier transform infrared spectroscopy (FT-IR) was measured using Film and KBr pellet method. Chemical bond groups present within NGO are visualized at relative wavelengths; O-H at 3426cm − 1 , C-H at 2920cm − 1 and 2853cm − 1 , C = O at 1731cm − 1 , C = C at 1629cm − 1 , C-OH at 1387cm − 1 and C-O at 1025cm − 1 (Fig. 3 C). Attenuated total reflectance fourier transform infrared spectroscopy (ATF-FT-IR) was also measured. C = O, -OH bending vibration and C = O were shown in 1733cm − 1 , 1635cm − 1 and 1630cm − 1 wavenumber, respectively and carbon single bonded hydrogen bending, and carbon single bonded oxygen stretching were located and marked at their established positions (Supple Fig. 2 B). Criteria for a compound to be labelled as nanoparticle, physical properties, such as size within the range of 1 to 100 nm and significantly larger surface area to volume ratio, and chemical properties, such as surface chemistry, must be met [ 18 ]. Both physical and chemical properties shown above using TEM, FT-IR and ATF-FT-IR clearly dictate NGO as nanomaterial. Effect of NGO treatment on PSEN1 AD cerebral organoids As shown above, phenotypic markers of AD are expressed at a higher level at Day 84; therefore, we added NGO to the PSEN1 AD brain organoids at Day 84 for a 2-week period (Fig. 4 A). NGO was administered at a concentration of 10 µg/ml, and when treated for a 2-week period, the organoids seemed to be covered by NGO (Supple Fig. 3 A). To prove that NGO successfully penetrated into the organoid, we used biotin-tagged NGO, and immunostaining images of biotin confirmed that NGO could passively diffuse into the brain organoids (Fig. 4 B). The NGO-treated group also did not show any cytotoxic phenotypes compared to the nontreated group. The level of c-cas3 expression did not differ in all groups (Fig. 4 C), and the area of the NGO-treated organoids did not differ compared to that of the nontreated organoids at Day 98 (Supple Fig. 3 A). In terms of neural development, immunostaining images of TUJ1, SOX2, MAP2 and KI67 showed no difference between the treated and nontreated groups within WT-CMC3 and PSEN1 AD brain organoids (Supple Fig. 3 B). Furthermore, this finding was supported by western blot images and quantified levels of Tuj1 and NeuN protein expression, as there were no significant differences between the treated and nontreated groups within WT-CMC3 and PSEN1 AD brain organoids (Supple Fig. 3 C). In summary, NGO treatment did not cause any cytotoxic activity or affect the development of brain organoids. As expected, there were changes in the expression level of AD phenotypes. In the case of pTau expression, both the treated and nontreated groups of PSEN1 AD brain organoids showed pTau expression. However, the level of Aβ secretion significantly decreased in the NGO-treated group compared to the nontreated group. This result was evident in both immunostaining images and quantified Aβ expression (Fig. 4 D). ELISA data also showed that the ratio of Aβ42/Aβ40 in the NGO-treated group decreased compared to that in the nontreated group (Fig. 4 E). Furthermore, western blot analysis of Aβ showed decreased expression of Aβ in the NGO-treated group compared to the nontreated group (Fig. 4 F). These data confirm that NGO can decrease Aβ secretion levels in PSEN1 AD brain organoids. NGO reduces Aβ levels via an autophagic mechanism To further investigate NGO’s mechanism of action on AD brain organoids, we examined multiple mechanisms by which NGO decreased Aβ levels. Li et al. have shown that graphene oxide could have an effect on inducing autophagy on a 2D cellular level [ 19 ] Therefore, we wanted to check if our NGO could also induce autophagy on human cerebral organoid. We screened for a list of proteins related to autophagy using western blotting analysis and found an increase in the level of Beclin1 in the NGO-treated group. Furthermore, in combination with the decrease in the level of p62 in the NGO-treated group, NGO treatment at a concentration of 10 µg/ml for a 2-week period activated the autophagic mechanism (Fig. 5 A, B). Furthermore, we examined the expression levels of LC3b, mTOR and AMPK. As we examined the phosphorylated forms of both mTOR and AMPK, NGO treatment elevated the ratio of p-AMPK expression to AMPK expression and decreased the ratio of p-mTOR to mTOR expression (Fig. 5 C, D). These outcomes indicate that NGO treatment inhibits mTOR activation by activating the AMPK signaling pathway in PSEN1 AD brain organoids. AMPK can be activated by two mechanisms: direct activation via an increase in ROS levels by modifying the AMPKα subunit and indirect activation via an increase in AMP and ADP levels [ 20 ]. To examine the detailed mechanism of NGO, we examined AMP, ADP and ATP levels in the NGO-treated and untreated PSEN1 AD brain organoids. As positive control, a potent AMPK indirect activator, A-769962, was employed. 2D induced neural stem cells (iNSCs) have been utilized in finding the optimal concentration of A-769962. iNSC is a reprogrammed cell line obtained from human fibroblast, as described in our previous study [ 10 ]. The optimal concentration was shown to be 100 µM after a cytotoxicity assay performed on 2D iNSCs (Fig. 5 E). In order to measure the effectiveness of NGO activating AMPK pathway, AMP/ATP ratio is required. This value was obtained using two different analysis tools: ADP/ATP ratio assay kit and AMP assay kit. Then by utilizing adenylate kinase equilibrium equation, AMP/ATP ratio was calculated. The NGO-treated group showed an increased AMP/ATP ratio compared to the control group; however, this ratio was slightly lower than that of A-769962 (Fig. 5 F). Activation of AMPK by NGO was also verified by measuring sirtuin 2 (SIRT2) level and ATG7. SIRT2 is known to enhance AMPK activation by deacetylating LKB1, which in turn promotes ATG7-mediated autophagosome formation. Both genes were upregulated in NGO treated group, which indicates an increased level of both SIRT2 and ATG7 promotes AMPK activation (Fig. 5 G). Gene expression analysis of WT and PSEN1 AD cerebral organoids As mentioned above, the phenotypical hallmark of AD is well known to be the aggregation of Aβ [ 3 , 4 ]. Even though treatment of NGO increases autophagy activity within the organoid, which then leads to removal of aggregated Aβ, however, this alone does not provide clear explanation on decrease in Aβ. Therefore, we utilized whole-transcriptome RNA sequencing to screen and identify gene expression differences between WT and PSEN1 AD cerebral organoids and also to discover any other therapeutic effect of NGO treatment AD cerebral organoids. RNA samples: WT-CMC3, NGO treated WT-CMC3, PSEN1 AD and NGO treated PSEN1 AD organoids at d98, were used to identify genes that were differentially expressed between different groups with or without NGO treatment. After 14 weeks of culture of 4 groups, diversifications were observed between all groups (Fig. 6 A). mRNA sequencing of four groups resulted in identifying around 43,000 genes, of which there were around 8,500 genes that showed statistically significant differences between groups. From the list of statistically significant differential gene expressions, the top 550 genes were selected for gene ontology analysis, including biological processes (BP), cellular components (CC), and molecular function (MF) (Fig. 6 B). Enrichment scores in all three ontologies illustrated an increase in multiple areas; but especially several sections were highly correlated with neural inflammation. For further simplification of illustrated gene ontology results, data were then categorized and linked with related genes and then visualized using Cnet plot of enriched KEGG pathways (Fig. 6 C) and then transformed into a bar graph (Fig. 6 D). KEGG pathway analysis discovered significant enrichments in areas correlated with inflammatory cytokines and their mechanisms, such as, cytokine-cytokine receptor interaction, TLR signaling pathway and NF-kappa B signaling pathway. Pathway analysis also indicated differential gene expression also linked with IL-17 pathway, which could implicate the relationship on NGO working within IL-17 signaling cascade. RNA-sequencing analysis once again illustrated the relationship between NGO treatment and neural inflammation, which then allowed us to further investigate the detailed mechanism of NGO on Aβ reduction. NGO reduces the level of newly formed Aβ by reducing proinflammatory cytokines As stated above, NGO activates autophagy by activating the AMPK signaling pathway, which then leads to the removal of secreted Aβ in PSEN1 AD brain organoids. Then, we examined whether NGO negatively participates in the formation of Aβ. NGO is known to have both proinflammatory and anti-inflammatory effects depending on the concentration and type of NGO [ 21 ]. Furthermore, KEGG pathway analysis also illustrated that inflammation related pathways were enriched; thus, we examined how NGO affects inflammation within AD brain organoids. To validate the inflammatory response of NGO on PSEN1 AD brain organoids, we examined the expression levels of both pro- and anti-inflammatory cytokines via RT‒PCR. In a comparison of the NGO-treated group and the nontreated group, there was a decrease in the expression of the proinflammatory cytokines TNF and IL-1A, whereas there was no difference between the groups in the anti-inflammatory cytokines IL-10 and TGFβ (Fig. 7 A). The presence of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (IBA1) expression in both groups indicates that astrocytes and microglial cells are expressed in cerebral brain organoids (Fig. 7 B). Microglial cells are known to express inducible nitric oxide synthase (iNOS) when in response to inflammatory stimuli. However, the decrease in pro-inflammatory cytokines, such as TNF and IL-1A, due to NGO treatment, lowers the activation of microglia and astrocytes, which then leads to lower iNOS expression. Also, since TNF acts on microglia and astrocytes to induce C3, lowered expression of TNF leads to lower expression of C3 in NGO treated group compared than control (Fig. 7 B). Due to the decrease in proinflammatory cytokine levels and decrease in the activation of astrocytes and microglial cells, it has been shown that the level of interferon-induced transmembrane protein 3 (IFITM3) expression also decreased in the NGO-treated group by immunostaining (Fig. 7 C). A decrease in IFITM3 levels also supported western blotting (Fig. 7 D). Also, as shown in the RNA sequencing data, we looked further into the NFkB signaling pathway. The decrease in the activation of NFkB alongside stable ERK1/2 expression but decrease in COX2 and IL-1b expression (Fig. 7 D) illustrates that NGO inhibits NFkB activity by increasing the expression of NFkb inhibitor beta (NFkBIB). In summary, NGO treatment decreased Aβ levels in PSEN1 AD brain organoids by decreasing NFkBIB and proinflammatory cytokines, which then led to inhibition of γ-secretase activity via a decrease in IFITM3 expression. Validation of NGO treatment on different patient-derived AD brain organoids To examine whether NGO could be a new therapeutical agent in treating AD or it is only suitable for PSEN1 A246E mutated patients, we obtained three more iPSCs; one WT iPSC cell line and two AD patient-derived iPSCs, for NGO validation. WT-Maru was obtained from Maru Therapeutics, wild-type iPSC that has different culturing conditions compared to WT-CMC3. Also, for AD patient-derived iPSCs, UCSD241i-APP2-3 (241AD) and CSD234i-SAD2-3 (234AD) were obtained from National Stem Cell Bank of Korea. 241AD iPSC is from a familial Alzheimer’s disease patient, similar to PSEN1 AD iPSCs, with an APP duplication genetical mutation and 234AD iPSC is from a sporadic Alzheimer’s disease patient diagnosed at the age of 78. Cerebral brain organoids were generated using the same protocol as above and each group; WT-Maru, 241AD and 234AD, were sampled at d42, d84 and d98 for analysis. These organoids were treated using the same NGO at identical concentration, used with WT-CMC3 and PSEN1 AD brain organoids. Sampled organoids at d42 and d84 were examined via immunohistochemistry, illustrating cell proliferation marker KI-67, neuronal progenitor marker SOX2, and neuronal markers TUJ1 and MAP2 (Fig. 8 A). Organoids from all three groups showed clear rosette formation along with clear neuronal development. As these cerebral brain organoids exhibited neuronal development, we then examined for the hallmarks of AD. As shown in Fig. 8 b, WT-Maru did not express Aβ nor p-Tau. On the other hand, 241AD and 234AD both showed formation of Aβ and p-Tau expression. WT-Maru, 241AD and 234AD brain organoids were also cultured for 12 weeks and then followed by 2 weeks treatment with NGO. Similar to WT-CMC3 and PSEN1 AD brain organoids, the neuronal markers, such as NeuN and Tuj1 expressions, were not affected in all groups (Fig. 8 C). In terms of Aβ expression, both 241AD and 234AD showed decrease in Aβ expression, as well as in Aβ-42 over Aβ-40 ratio, there is an obvious decrease in both AD patient-derived brain organoids (Fig. 8 C,D). However, the mechanism of action behind the decreased expression was slightly different. Western blotting images clearly illustrates the obvious increase of beclin1 expression followed by decreased expression of p62 in NGO treated groups, as well as decrease in the ratio of LC3BII to LC3BI clearly indicates that NGO facilitates autophagy mechanism and aid in removing aggregated Aβ. In case of 241AD brain organoids, AMPK activation mechanism of action is very similar to PSEN1 AD brain organoids, as the ratio of p-AMPK/AMPK is significantly higher in NGO treated group than control group in 241AD. However, even though there is a slight increase of p-AMPK/AMPK ratio in 234AD, the statistical analysis shows that the difference is not significant (Fig. 8 C). This similar outcome is also shown in inflammatory response mechanism. Starting with decreased expression of COX2 and IL-1b in NGO treated group is similar to PSEN1 AD brain organoid for both 241AD and 234AD organoids. However, even if ERK1/2 level is not changed, the activation of NFkB only occurred in 241AD brain organoids and not in 234AD brain organoids. Similarly, the difference is also found in the IFITM3 expression level. The protein expression level graph illustrates the change between control and NGO treated group in 234AD, but the change is not statistically significant (Fig. 8 C). DISCUSSION AD is a neurodegenerative disorder characterized by progressive cognitive decline and memory loss [ 22 ]. One of the defining phenotypes of AD is the formation and accumulation of Aβ in the brain. The build-up of Aβ is accompanied by the development of neurofibrillary tangles, which result from the phosphorylation of the Tau protein within nerve fibers. The presence of these neurofibrillary tangles and Aβ deposits leads to the degeneration and loss of neurons in the brain and exacerbates other factors associated with AD, such as inflammation, oxidative stress, and neural microvascular disease [ 1 , 3 , 21 ]. Despite the major impact of AD on cognitive function, there are currently no treatments that have been proven to be both safe and effective [ 4 , 5 ]. This issue is partly due to the limitations of existing animal models and 2D cell culture systems, which do not fully replicate the specific symptoms and indications observed in patients with AD [ 6 , 7 ]. The expression of multiple familial Alzheimer's disease (FAD) mutations in animal models accelerates pathological markers that resemble human symptoms. However, despite promising therapeutic effects observed in these models, no treatments have yet demonstrated significant breakthroughs in human clinical trials [ 23 ]. Drummond et al. further highlights that the low success rate—reported to be as high as 99.6% failure—in Alzheimer’s disease clinical trials underscores the premature translation of pathology reduction in transgenic mice to human applications. To address these limitations, our research aims to confirm that a human cerebral brain organoid model generated from AD patient-derived iPSCs can accurately reproduce the hallmark features of AD. This model can then be used as a platform for testing the efficacy of potential drugs for AD treatment. This approach builds on previous research that has used 3D organoids to model disease in multiple organs, including the liver, brain, and intestine, and the use of brain organoids to study other neurodegenerative diseases such as Parkinson's disease, microcephaly, and amyotrophic lateral sclerosis [ 6 , 7 ]. As shown above, we successfully generated AD cerebral brain organoids using patient-derived iPSCs. After culturing these organoids for an extended period of time, they accurately mimicked both hallmarks of AD phenotypes. Thus, we were able to examine the impact of NGO on AD cerebral brain organoids utilizing this successful high-throughput drug testing platform. Biotin-tagged NGO showed that the size of NGO was an advantage, as it could passively penetrate into the brain organoids without any external force applied. The application of NGO did not physically damage the organoids or cause any neurodegenerative effects. However, when applied at a specific concentration, NGO exerted its therapeutic effects and decreased the level of Aβ by activating multiple signaling pathways. The presence of astrocytes and microglia in this cerebral brain organoid model was the key to investigating the inflammatory effect of NGO. Generally, neuroinflammation occurs from microglial activation from increase in proinflammatory cytokines. Proinflammatory cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), bind to receptors in cells and activate signaling pathways that lead to the transcriptional activation of the iNOS gene. Leading to upregulation of C3 and iNOS, cause oxidative stress, mitochondrial dysfunction and potential for neurodegeneration [ 24 ]. In response to the decrease in proinflammatory cytokine levels by NGO treatment, the activation of these signaling pathways will also decrease, leading to a decrease in C3 and iNOS expression and production of nitric oxide (NO). This process can contribute to a reduction in the levels of inflammation in the tissue. These factors had an impact on the reduced IFITM3 secretion in the group treated with NGO. Hur et al. stated that IFITM3 is a γ-secretase modulatory protein that increases inflammatory cytokines and stimulates the expression of IFITM3 in astrocytes [ 25 ]. In the NGO-treated group, this effect led to diminished activity of γ-secretase, thus decreasing Aβ formation. The activation of AMPK increases autophagy, the process of cellular degradation and recycling [ 26 ]. This activation supports the formation of autophagosomes and enhances lysosomal degradation. This process is believed to maintain cellular stability and protect cells from stress factors. In contrast, mTOR, a regulator of cellular metabolism and growth, obstructs autophagy by blocking autophagosome formation, but AMPK can deactivate mTOR to increase autophagy [ 27 ]. An increase in beclin1 is a critical component of the autophagic initiation complex and helps promote autophagosome formation [ 28 ]. A reduction in the levels of p62, which acts as a scaffold protein in autophagic regulation, releases autophagy-related proteins and activates autophagy. A decrease in the LC3B II/LC3B I ratio is a sign of increased autophagy, as LC3B is involved in autophagic regulation. In terms of AMPK activation by NGO, the exact mechanism by which NGO increases the levels of AMP and ADP is not well understood. However, it is likely that NGO affects cellular energy metabolism, which can lead to changes in the levels of AMP and ADP [ 27 ]. In general, AMP and ADP levels increase when cellular energy stores are depleted, and this increase is sensed by AMPK, which is activated in response [ 30 , 31 ]. NGO may affect cellular energy metabolism through oxidative stress or inflammation, which can lead to changes in the levels of AMP and ADP [ 32 , 33 ]. However, more research is needed to fully understand the exact mechanism by which NGO affects AMP and ADP levels. The effect of NGO treatment was further evaluated using two more AD patient-derived iPSCs; CSD234i-SAD2-3 (234AD) and USCD241i-APP2-3 (241AD). 234AD iPSC is from a sporadic AD patient, who has been diagnosed with AD at the age of 78 and cells were collected at the age of 83, and 241AD iPSC is from a familial AD patient with APP duplication genetic alteration [ 34 ]. Treatment of NGO onto different AD brain organoids also showed similar growth compared to WT brain organoids without any neurodegenerative signals. However, the effect of NGO on each type of AD were both similar and different in some areas. As illustrated above, both 241AD and 234AD showed decrease in Aβ levels, showing promising effect of NGO. However, even though there were slight decrease in the ratio of p-AMPK/AMPK and p-NFkB/NFkB, the level of drop was not shown to be significant. 241AD showed similar effect as PSEN1 AD brain organoids as they are both from Familial Alzheimer's disease (FAD) patients, however, 234AD from sporadic Alzheimer's disease (SAD) patient did not. FAD and SAD exhibit similar underlying pathology and disease progression. However, they differ in genetic etiology and age of onset. FAD is inherited in an autosomal dominant manner and typically manifests earlier in life, whereas SAD, the more prevalent form, has a later onset and results from a complex interaction of genetic predisposition and environmental factors [ 35 ]. Although NGO exhibits therapeutic effects by reducing Aβ levels through modulation of both autophagy and neuroinflammatory pathways, the variability in genetic and environmental factors in SAD patient likely contributes to the observed differences in treatment outcomes [ 34 ]. To better understand the influence of individual patient-specific factors on therapeutic efficacy, it is essential to conduct systematic screenings utilizing a series of iPSC lines derived from both FAD and SAD patients. Even though this study specifically illustrates the therapeutical actions of NGO towards AD brain organoid, it also enlightens some limitations that should be addressed. First, due to the fundamental design of an organoid culture system, this model does not have an excretion mechanism. Therefore, the long-term effect of NGOs within the system needs to be studied. Furthermore, even though organoid models show a higher level of similarity to patients than in vivo models, the effect of NGO treatments on an AD mouse model is needed. Since brain organoid model is not composed with blood-brain barrier system, in vivo models would be an option in exploring the excretion system as well as whether NGO could pass through the blood‒brain barrier and shows a similar effect compared to an organoid model. CONCLUSION In conclusion, this study suggests the therapeutical applications of NGO towards AD treatment by decreasing aggregation and generation of Aβ. Treatment of NGO on AD patient-derived brain organoid operating via multiple pathways, such as increasing neural autophagic activities by AMPK activation and reducing proinflammatory cytokines and IFITM3 expression via NFkB pathway, strongly illustrate the possibility in utilizing nano graphene oxides for treatment of neurodegenerative diseases. Abbreviations Alzheimer’s disease AD Amyloid precursor protein APP Amyloid-beta Aβ Atomic force microscopy AFM Attenuated total reflectance fourier transform infrared spectroscopy AFT-TF-IR Biological processes BP Bovine serum albumin BSA Cellular components CC Enhanced chemiluminescence ECL Familial AD FAD Fourier transform infrared spectroscopy FT-IR Glial fibrillary acidic protein GFAP Hyperphosphorylated tau pTau Induced neural stem cells iNSCs Induced pluripotent stem cells iPSCs Inducible nitric oxide synthase iNOS Interferon-induced transmembrane protein 3 IFITM3 Interleukin-1 IL-1 Interleukin-6 IL-6 Ionized calcium-binding adapter molecule 1 IBA1 Molecular function MF Nano-graphene oxide NGO Neurofibrillary tangles NFTs NFkb inhibitor beta NFkBIB Nitric oxide NO Presenilin-1 PSEN1 Sporadic AD SAD Transmission electron microscopy TEM Tumor necrosis factor-alpha TNF-α Declarations ETHICS APPROVAL AND CONSENT TO PARTICIPATE In terms of the iPSC lines used in this study, all iPS cell lines underwent ethics approval under the project title of “Therapeutic effect of deca nano-graphene oxide treatment on Alzheimer’s disease in the patient-derived Alzheimer’s disease brain organoids” by Seoul National University Institutional Bioethics Committee, IRB No. 2511/004-016 , 24 th June 2024. CS40iFAD-nxx (RRID:CVCL_YX94) cell line is available for purchase for both commercial and academic purposes at https://biomanufacturing.cedars-sinai.org/product/cs40ifad-nxx/. WT-Maru cell line can be provided upon request at https://marurx.com/contact-us/. Ethical approval of WT-Maru can be found at Public IRB number P01-202110-31-009 and published paper with related cells [37]. CMC-hiPSC-003 cell line can be provided upon request at https://nih.go.kr/ncsr/nscb/kr/cdc/scb/scbSalesInfo.do. Ethical approval of CMC-hiPSC-003 can be found at Rim et al. [38] and also at https://nih.go.kr/ncsr. Ethics approval of CSD234i-SAD2-3 and UCSD241i-APP2-3 can be found at Israel et al. [35]. CONSENT FOR PUBLICATION Not Applicable DATA AVAILABILITY STATEMENT All data supporting the conclusions of the paper are available in the article and corresponding figures. The sequencing data supporting the results reported in this study have been deposited in the NCBI GEO under the accession number GSE266055. Additional experimental details and more detailed data used or analyzed in this study are available from the corresponding author upon reasonable request. COMPETING INTERESTS The authors declare that they have no competing interests. FUNDING This research was supported by the Bio&Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. RS-2023-00266110). This study was partially supported by the Research Institute for Veterinary Science, Seoul National University. This study was partially supported by the Research Institute for Veterinary Science, Seoul National University. AUTHOR CONTRIBUTIONS In vitro experiments were performed by NGK. Partial iPSC cell lines have been provided by HP and HK. RNA, protein and organoid sampling were performed by NGK and JC. Data analysis was performed by NGK and JC. Data presentation was performed by NGK. RNA-seq was performed by NGK. NGO analysis was performed by NGK, SWC and JCR. Experimental design was performed by NGK and KSK. The manuscript was written by NGK and KSK. ACKNOWLEDGEMENTS The authors declare that they have not used AI-generated work in this manuscript. [김2] AUTHOR INFORMATION FIRST AUTHOR Nam Gyo Kim CORRESPONDING AUTHOR Kyung-Sun Kang AUTHORS AND AFFILIATIONS The Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea Nam Gyo Kim, Jaeyong Chun, Hyeyeon Park , Kyung-Sun Kang Division of Intractable Disease Research, National Institute of Health, Osong, Cheongju, 28160, Republic of Korea Hyeyeon Park, Hyunyoung Kim Institutes of Convergence Technology, INBCT Co.,LTD, Hwaseong-si 18462, Republic of Korea Soon Won Choi, Jae-Chul Ryu References Breijyeh Z, Karaman R. Comprehensive review on Alzheimer’s disease: causes and treatment. Molecules. 2020 Dec 8;25(24):5789. Lanoiselée HM, Nicolas G, Wallon D, Rovelet-Lecrux A, Lacour M, Rousseau S. APP, PSEN1, and PSEN2 mutations in early-onset Alzheimer disease: A genetic screening study of familial and sporadic cases. PLoS medicine. 2017 Mar 28;14(3):e1002270. Chen ZY, Zhang Y. Animal models of Alzheimer’s disease: Applications, evaluation, and perspectives. Zoological research. 2022 Nov 11;43(6):1026. Rajmohan R, Reddy PH. Amyloid-beta and phosphorylated tau accumulations cause abnormalities at synapses of Alzheimer’s disease neurons. Journal of Alzheimer's Disease. 2017 Jan 1;57(4):975-99. Hane FT, Robinson M, Lee BY, Bai O, Leonenko Z, Albert MS. Recent progress in Alzheimer’s disease research, part 3: diagnosis and treatment. Journal of Alzheimer's disease. 2017 Jan 1;57(3):645-65. Lovett ML, Nieland TJ, Dingle YT, Kaplan DL. Innovations in 3D tissue models of human brain physiology and diseases. Advanced functional materials. 2020 Oct;30(44):1909146. Kim J, Koo BK, Knoblich JA. "Human organoids: model systems for human biology and medicine." Nature Reviews Molecular Cell Biology 21.10 (2020): 571-584. Lee SE, Choi H, Shin N, Kong D, Kim NG, Kim HY. Zika virus infection accelerates Alzheimer’s disease phenotypes in brain organoids. Cell Death Discovery. 2022 Apr 2;8(1):153. Kook MG, Lee SE, Shin N, Kong D, Kim DH, Kim MS. Generation of cortical brain organoid with vascularization by assembling with vascular spheroid. International Journal of Stem Cells. 2022;15(1):85. Lee SE, Kwon D, Shin N, Kong D, Kim NG, Kim HY. Accumulation of APP-CTF induces mitophagy dysfunction in the iNSCs model of Alzheimer’s disease. Cell Death Discovery. 2022 Jan 10;8(1):1. Kong D, Park KH, Kim DH, Kim NG, Lee SE, Shin N, Kook MG, Kim YB, Kang KS. Cortical-blood vessel assembloids exhibit Alzheimer’s disease phenotypes by activating glia after SARS-CoV-2 infection. Cell Death Discovery. 2023 Jan; 9(32). Dawson TM, Golde TE, Lagier-Tourenne C. Animal models of neurodegenerative diseases. Nature neuroscience. 2018 Oct;21(10):1370-9. Whitby RL. Chemical control of graphene architecture: tailoring shape and properties. ACS nano. 2014 Oct 28;8(10):9733-54. Kim HY, Yoon HH, Seong H, Seo DK, Choi SW, Ryu J. Preventive effects of nano-graphene oxide against Parkinson’s disease via reactive oxygen species scavenging and anti-inflammation. BMB reports. 2023 Mar 3;56(3):202. Yu A, Park HS, Chae DH, Park JH, Heo J, Cho K, Kim J, Lee H, Jee S, Kim C, Choi SW, Ryu J, Hur EH, Choi Y, Choi EJ, Oh MK, Lee HY, Lee JH, Yu KR. Nanographene Oxide Attenuates Acute GVHD by Modulating Macrophage Polarization in a Xenogeneic Mouse Model. Advanced Science. 2025 Dec 4;12(45). Kim DH, Kim MJ, Kwak SY, Jeong J, Choi D, Choi SW. Bioengineered liver crosslinked with nano-graphene oxide enables efficient liver regeneration via MMP suppression and immunomodulation. Nature Communications. 2023 Feb 13;14(1):801. Lancaster MA, Knoblich JA. Generation of cerebral organoids from human pluripotent stem cells. Nature protocols. 2014 Oct;9(10):2329-40. EID, May M. Characterization of Nanoparticles by FTIR and FTIR-Microscopy. In: Handbook of consumer nanoproducts. Springer Singapore. 2022; p. 1-30. Li X, Li K, Chu F, Huang J, Yang Z. Graphene oxide enhances β-amyloid clearance by inducing autophagy of microglia and neurons. Chemico-Biological Interactions. 2020 Jul 1;325:109126. Kim J, Yang G, Kim Y, Kim J, Ha J. AMPK activators: mechanisms of action and physiological activities. Experimental & molecular medicine. 2016 Apr;48(4):e224-. Bałaban J, Wierzbicki M, Zielińska-Górska M, Sosnowska M, Daniluk K, Jaworski S. Graphene Oxide Decreases Pro-Inflammatory Proteins Production in Skeletal Muscle Cells Exposed to SARS-CoV-2 Spike Protein. Nanotechnology, Science and Applications. 2023 Dec 31:1-8. Jicha GA, Carr SA. Conceptual evolution in Alzheimer's disease: implications for understanding the clinical phenotype of progressive neurodegenerative disease. Journal of Alzheimer's Disease. 2010 Jan 1;19(1):253-72. Drummond E, Wisniewski T. Alzheimer’s disase: experimental models and reality. Acta Neuropathol. 2016 Dec 26;133(2):155-175. Zhang W, Xiao D, Mao Q, Xia H. Role of neuroinflammation in neurodegeneration development. Signal Tansduction and Targeted Therapy. 2023 Jul 12;267. Hur JY, Frost GR, Wu X, Crump C, Pan SJ, Wong E. The innate immunity protein IFITM3 modulates γ-secretase in Alzheimer’s disease. Nature. 2020 Oct 29;586(7831):735-40. Tamargo-Gómez I, Mariño G. AMPK: regulation of metabolic dynamics in the context of autophagy. International journal of molecular sciences. 2018 Nov 29;19(12):3812. Deleyto-Seldas N, Efeyan A. The mTOR–autophagy axis and the control of metabolism. Frontiers in cell and developmental biology. 2021 Jul 1;9:655731. Lin SC, Hardie DG. AMPK: sensing glucose as well as cellular energy status. Cell metabolism. 2018 Feb 6;27(2):299-313. Kang R, Zeh HJ, Lotze MT, Tang D. The beclin 1 network regulates autophagy and apoptosis. Cell Death & Differentiation. 2011 Feb 11;18(4):571-580. Gowans GJ, Hardie DG. AMPK: a cellular energy sensor primarily regulated by AMP. Biochemical Society Transactions. 2014 Jan 23;42(1):71-75. Hardie DG. AMP-activated protein kinase—an energy sensor that regulates all aspects of cell function. Genes & development. 2011 Sep 15;25(18):1895-908. Camacho-Castillo L, Phillips-Farfán BV, Rosas-Mendoza G, Baires-Lopez A, Toral-Rios D, Campos-Pena V. Increased oxidative stress contributes to enhance brain amyloidogenesis and blunts energy metabolism in sucrose-fed rat: Effect of AMPK activation. Scientific Reports. 2021 Oct 1;11(1):19547. Masenga SK, Kabwe LS, Chakulya M, Kirabo A. Mechanisms of oxidative stress in metabolic syndrome. International journal of molecular sciences. 2023 Apr 26;24(9):7898. Israel MA, Yuan SH, Bardy C, Reyna SM, Mu Y, Herrera C, Hefferan MP, Van Gorp S, Nazor KL, Boscolo SF, Carson CT, Laurent LC, Marsala M, Gage FH, Remes AM, Koo EH, Goldstein LS. Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells. Nature, 2012; 482(7384), 216-220. Piaceri I, Nacmias B, Sorbi S. Genetics of familial and sporadic Alzheimer’s disease. Frontiers in Bioscience-Elite, 2023 Jan 1;5(1):167-177. Quan M, Cao S, Wang Q, Wang S, Jia J. Genetic phenotypes of Alzheimer’s disease: Mechanisms and potential therapy. Phenomics, 2023 Apr 3;3(4):333-349. Kwon D, Moon BK, Han M, Lee TW, Lee J, Kang KS. Genetically stable multi-gene edited iPSCs-derived NK cells for enhanced cancer immunotherapy. Molecular Therapy Oncology, 2024; 32.4. Rim TA, Park N, Nam Y, Ham DS, Kim JW, Ha HY, Jung JW, Jung SM, Baek IC, Kim SY, Kim TG, Song J, Lee J, Park SH, Chung NG, Yoon KH, Ju JH. Recent progress of national banking project on homozygous HLA‐typed induced pluripotent stem cells in South Korea. Journal of tissue engineering and regenerative medicine. 2018 Dec 3; e1531-e1536. Tang D, Chen M, Huang X, Zhang G, Zeng L, Zhang G. SRplot: A free online platform for data visualization and graphing. PLoS One. 2023 Nov 9;18(11):e0294236. Additional Declarations No competing interests reported. Supplementary Files daNGOParticleSizeAnalysis.csv GOFigureSupplement1.jpg SupplementalFigure 1. Brain Organoid Generation A) Representative image of WT-CMC3, WT-Maru, PSEN1 AD, 241AD and 234AD cerebral brain organoid generation at different stages. Scale bars showing 1mm. B) Average growth of cerebral brain organoids measured in terms of area at different stages. The results are presented as the average±SD (n=10). GOFigureSupplement2.jpg Supplemental Figure 2. NGO Characterization A) Particle lateral size measurements shown as a distribution graph, showing average value of 10nm and 99.9% distribution between 5nm to 33.7nm. A total of 2.8x10 12 particles were measured, measured raw data is presented in Table 1. B) Attenuated total reflectance fourier transform infrared spectroscopy (ATF-FT-IR) graph, each arrow and box representing peak wavelength of designated chemical bonds. GOFigureSupplement3.jpg Supplemental Figure 3. No Effect of NGO on Brain Organoid Neurogenesis A) Representative image of PSEN1 cerebral brain organoids and average area calculation at d84 and d98 for NGO non-treated and treated group. Scale bars showing 1cm. Statistical analysis was performed by Student’s t-test. The results are presented as the average±SD (n=10). B) Representative images of immunostaining of WT and PSEN1 cerebral brain organoids at d98 with or without NGO treatment; neuronal markers (TUJ1, MAP2), neuroepithelium marker (SOX2), proliferation marker (Ki67). Nuclei were stained with DAPI. Scale bars showing 100μm. Statistical analysis was performed by Student’s t-test. The results are presented as the average±SD (n=6). C) Amount of neuronal marker (TUJ1 and NeuN) expressions were visualized using western blotting technique and quantified using ImageJ. Statistical analysis was performed by Student’s t-test. The results are presented as the average±SD (n=3, 3 organoids per sample, total of 9 organoids were used). Unedited versions of all western blot images are shown and labelled in supplementary figure 4. GOFigureSupplement4a.jpg Supplemental Figure 4. Full Length Western Blots A) Unedited images of western blots in figure 4F. GOFigureSupplement4b.jpg B) Unedited images of western blots in figure 5a. GOFigureSupplement4c.jpg C) Unedited images of western blots in figure 5c. GOFigureSupplement4d.jpg D) Unedited images of western blots in figure 7d. E) Unedited images of western blots in figure 5c. D) Unedited images of western blots in figure 8c. F) Unedited images of western blots in supplementary figure 3c. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8383146","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":594910054,"identity":"fef437f3-d1ae-4338-a8bb-deafc4b2a0f0","order_by":0,"name":"Nam Gyo Kim","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Nam","middleName":"Gyo","lastName":"Kim","suffix":""},{"id":594910055,"identity":"37189fdb-5be8-4245-b6cd-a7de207f9053","order_by":1,"name":"Jaeyong Chun","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Jaeyong","middleName":"","lastName":"Chun","suffix":""},{"id":594910058,"identity":"72c625de-c8f6-4dde-ad59-7a8c652603d1","order_by":2,"name":"Hyeyeon Park","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Hyeyeon","middleName":"","lastName":"Park","suffix":""},{"id":594910060,"identity":"43e97e06-6659-4178-b902-d330631966a0","order_by":3,"name":"Hyunyoung Kim","email":"","orcid":"","institution":"National Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Hyunyoung","middleName":"","lastName":"Kim","suffix":""},{"id":594910062,"identity":"63efa32a-583c-4e26-8589-bb4c5972f71d","order_by":4,"name":"Soon Won Choi","email":"","orcid":"","institution":"Institute of Bio \u0026 Nano Convergence, INBCT Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Soon","middleName":"Won","lastName":"Choi","suffix":""},{"id":594910063,"identity":"c4313b97-3757-4441-903e-33ae673ea969","order_by":5,"name":"Jaechul Ryu","email":"","orcid":"","institution":"Institute of Bio \u0026 Nano Convergence, INBCT Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Jaechul","middleName":"","lastName":"Ryu","suffix":""},{"id":594910064,"identity":"d129f1c1-85da-4cf8-a329-3568dda9d261","order_by":6,"name":"Kyung-Sun Kang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYBACCQh1QA4mYEC0FmPStSQ2EK1Fsv3s4deFbXfS58/uMWD4UcNgbN5AQIs0T16a9cy2Z7kb7pwxYOw5xmAmc4CAFjmGHDNj3rbDuRskcgwYeBsYbCQIOUyO/w1YS7r8jBwDxr/EaJGWyDF+DNSSwHAjx4AZaIsZQS2SM96YMc8498xww420gsMyxySMCWqROJ9j/Lmg7I68/IzkjQ/f1NgYziCkBQjYpGGsA/B4IgCYPxOlbBSMglEwCkYuAADqozvAH4cFGwAAAABJRU5ErkJggg==","orcid":"","institution":"Seoul National University","correspondingAuthor":true,"prefix":"","firstName":"Kyung-Sun","middleName":"","lastName":"Kang","suffix":""}],"badges":[],"createdAt":"2025-12-17 08:08:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8383146/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8383146/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103338630,"identity":"243b3e27-d097-4448-81d2-5cb1a509ee5d","added_by":"auto","created_at":"2026-02-24 15:07:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":611858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eWT-CMC3 and PSEN1 Patient-Derived AD Brain Organoid Generation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA) Graphic diagram of the basic information about PSEN1-mutated AD patient-derived iPSCs. B) Schematic image illustrating the timeline of iPSC-derived cerebral brain organoids. C) Representative images of immunostaining of WT-CMC3 and PSEN1 cerebral brain organoids at d42 and d84; neuronal markers (TUJ1, MAP2), neuroepithelium marker (SOX2), proliferation marker (Ki67). Nuclei were stained with DAPI. Scale bars showing 100μm. D) Quantification of cerebral organoid related marker expressions using ImageJ. Statistical analysis was performed by Student’s t-test. The results are presented as the average±SD (n=7).\u003c/p\u003e","description":"","filename":"GOFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/3a65189969dc3d7eebcbbecb.jpg"},{"id":103506740,"identity":"a7de6d1e-8b95-4f75-bb6a-52c875788a47","added_by":"auto","created_at":"2026-02-26 13:39:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":584205,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePSEN1 Brain OrganoidsExpressing the AD Phenotype\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA) Graphic diagram of the basic information about PSEN1 gene mutation of patient-derived iPSC, illustrating the impairment of γ-secretase and the formation of Aβ. B) Representative images of immunostaining of WT and PSEN1 cerebral brain organoids at d42 and d84; Alzheimer’s disease related markers (Tau, p-Tau and Aβ). Nuclei were stained with DAPI. Scale bars showing 100μm. The red-dashed box illustrates the magnified area. C) Quantification of cerebral organoid related marker expressions using ImageJ. Statistical analysis was performed by Student’s t-test; *P \u0026lt; 0.05, ****P \u0026lt; 0.0001. The results are presented as the average±SD (n=3).\u003c/p\u003e","description":"","filename":"GOFigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/8802e27b0a3c96285a6ce21c.jpg"},{"id":103506376,"identity":"f8fc4770-966a-49e0-9d6b-9998a473f4c8","added_by":"auto","created_at":"2026-02-26 13:35:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":384256,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eNGO Characterization\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA) Representative AFM image of NGO, showing the height of each particle at 0.72nm and 0.77nm. B) Representative TEM image of NGO. Scale bars showing 20nm. C) FT-IR graph measured using Film and Kbr pellet method, each arrow representing peak wavelength of designated chemical bonds.\u003c/p\u003e","description":"","filename":"GOFigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/70e59d01f61e76e782a758c6.jpg"},{"id":103338635,"identity":"5612fb6e-251c-46f2-bbc4-8618a7ee3b82","added_by":"auto","created_at":"2026-02-24 15:07:01","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":758299,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEffect of NGO treatment on PSEN1 AD organoids\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA) Schematic image illustrating the timeline of iPSC-derived cerebral brain organoids NGO treatment. B) Representative images of immunostaining of WT and PSEN1 cerebral brain organoids at d98 after NGO-biotin treatment. Nuclei were stained with DAPI. Scale bars showing 100μm. C) Representative images of immunostaining of WT and PSEN1 cerebral brain organoids at d98 with or without NGO treatment; cellular apoptotic marker (c-cas3). Nuclei were stained with DAPI. Scale bars showing 100μm. Quantification of c-cas3 expression using ImageJ. Statistical analysis was performed by Student’s t-test. The results are presented as the average±SD (n=3). D) Representative images of immunostaining of WT-CMC3 and PSEN1 cerebral brain organoids at d98 with or without NGO treatment; Alzheimer's disease related markers; Tau, p-Tau, Aβ. Nuclei were stained with DAPI. Scale bars showing 100μm. Quantification of Aβ using ImageJ. Statistical analysis was performed by Student’s t-test, **P \u0026lt; 0.01. The results are presented as the average±SD (n=4). E) Lysates of WT-CMC3 and PSEN1 cerebral brain organoids were analyzed by ELISA at d98. The amount of Aβ1-40 and Aβ1-42 in the RIPA fraction were measured using Aβ42/Aβ40 ELISA KIT (n=3, 3 organoids per sample, total of 9 organoids were used). F) Amount of Aβ expressions were visualized using western blotting technique and quantified using ImageJ (n=3, 3 organoids per sample, total of 9 organoids were used). Unedited versions of all western blot images are shown and labelled in supplementary figure 4.\u003c/p\u003e","description":"","filename":"GOFigure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/746d344042f03c114d60191c.jpg"},{"id":103506910,"identity":"f39e8d53-d070-4038-b846-86d42c2ad8e3","added_by":"auto","created_at":"2026-02-26 13:39:53","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":390724,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eThe AD treatment effect of NGO is dependent on the autophagic mechanism\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA) Autophagy related marker expressions; Beclin1 and p62, were measured using western blotting technique and B) quantified using ImageJ. Statistical analysis was performed by Student’s t-test, *P \u0026lt; 0.05. The results are presented as the average±SD (n=3, 3 organoids per sample, total of 9 organoids were used). C) Autophagy related marker LC3b expression and relative mTOR and AMPK activation against phosphorylated versions are visualized using western blotting technique and D) quantified using ImageJ. Statistical analysis was performed by Student’s t-test, *P \u0026lt; 0.05. The results are presented as the average±SD (n=3, 3 organoids per sample, total of 9 organoids were used). E) Cell viability was assessed in the presence of A-769662 (1-500μM). F) Normalized AMP/ATP ratio was calculated from ADP/ATP ratio assay and AMP assay. Statistical analysis was performed by Student’s t-test, *P \u0026lt; 0.05. The results are presented as the average±SD (n=3, 3 organoids per sample, total of 9 organoids were used). G) mRNA expression of autophagy related markers; SIRT2 and ATG7, in WT-CMC3, PSEN1 NGO non-treated and treated groups were measured using qRT-PCR. Statistical analysis was performed by Student’s t-test, *P \u0026lt; 0.05. The results are presented as the average±SD (n=3, 3 organoids per sample, total of 9 organoids were used). Unedited versions of all western blot images are shown and labelled in supplementary figure 4.\u003c/p\u003e","description":"","filename":"GOFigure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/9dab69338b46b5dbb6d462c5.jpg"},{"id":103506683,"identity":"fb3e3297-7f9f-4e76-826d-a6ae124ccb5b","added_by":"auto","created_at":"2026-02-26 13:38:56","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":668441,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eGene expression analysis of WT-CMC3 and PSEN1 AD organoids\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA) Differentially expressed genes in WT-CMC3 and PSEN1 AD organoids at d98. Z-scores of RNA-sequencing data of WT NGO non-treated and treated groups and PSEN1 NGO non-treated and treated groups on d98 were expressed as heat maps. Each sample was prepared using three organoids in all groups. B) Gene ontology results on three different ontologies; BP, CC, MF, of the top 550 genes that were differentially regulated. C) Cnet plot of enriched KEGG pathways. D) KEGG pathways analysis enrichment scores of four groups.\u003c/p\u003e","description":"","filename":"GOFigure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/4e43a909373e288ca2ba750a.jpg"},{"id":103338644,"identity":"1d98fb2a-0ac6-499e-bd82-39a9f0bd11ef","added_by":"auto","created_at":"2026-02-24 15:07:02","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":694831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEffect of NGO on the newly formed Ab via inflammation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA) mRNA expression of pro- and anti-inflammatory cytokine related markers; TNF, IL-1a, IL-10 and IL-1b, in PSEN1 NGO non-treated and treated groups were measured using qRT-PCR. Statistical analysis was performed by Student’s t-test, *P \u0026lt; 0.05, ***P\u0026lt;0.001. The results are presented as the average±SD (n=3, 3 organoids per sample, total of 9 organoids were used). B) Representative images of immunostaining of PSEN1 NGO non-treated and treated organoids at d98 for astrocyte marker (GFAP), microglial cell marker (IBA-1), and inflammation markers (C3 and iNOS). Nuclei were stained with DAPI. Scale bars showing 100μm. C) Representative images of immunostaining of PSEN1 NGO non-treated and treated organoids at d98 for IFITM3. Nuclei were stained with DAPI. Scale bars showing 100μm. Quantification was done using ImageJ. Statistical analysis was performed by Student’s t-test, **P \u0026lt; 0.01. The results are presented as the average±SD (n=6). D) Amount of IFITM3 expressions were visualized using western blotting technique and quantified using ImageJ. Statistical analysis was performed by Student’s t-test, **P \u0026lt; 0.01. The results are presented as the average±SD (n=3, 3 organoids per sample, total of 9 organoids were used). And amount of inflammation related markers (COX2, IL-1b) NFkB, p-NFkB and ERK1/2) expressions were visualized using western blotting technique and quantified using ImageJ. Statistical analysis was performed by Student’s t-test, *P \u0026lt; 0.05, **P \u0026lt; 0.01. The results are presented as the average±SD (n=3, 3 organoids per sample, total of 9 organoids were used). E) mRNA expression of NFkBIB in PSEN1 NGO non-treated and treated groups were measured using qRT-PCR. Statistical analysis was performed by Student’s t-test, **P \u0026lt; 0.01. The results are presented as the average±SD (n=3, 3 organoids per sample, total of 9 organoids were used). Unedited versions of all western blot images are shown and labelled in supplementary figure 4.\u003c/p\u003e","description":"","filename":"GOFigure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/6a812162418ebecce90303cf.jpg"},{"id":103338640,"identity":"ab8c9681-bd0f-4f70-9d5c-5d1c9ba7bdf2","added_by":"auto","created_at":"2026-02-24 15:07:01","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":192853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eValidation of NGO treatment on different Patient Derived AD Brain Organoids\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA) Representative images of immunostaining of WT-Maru, 241AD and 234AD cerebral brain organoids at d42 and d84; neuronal markers (TUJ1, MAP2), neuroepithelium marker (SOX2), proliferation marker (Ki67). Nuclei were stained with DAPI. Scale bars showing 100μm. B) Representative images of immunostaining of WT and PSEN1 cerebral brain organoids at d42 and d84; Alzheimer’s disease related markers (Tau, p-Tau and Aβ). Nuclei were stained with DAPI. Scale bars showing 100μm. The red-dashed box illustrates the magnified area. C) Autophagy related marker expressions; Beclin1 and p62, inflammation related marker expressions; IFITM3, COX2, IL-1b, NFkB, p-NFkB and ERK1/2, neuronal marker expression; Tuj1 and NeuN, and Aβ expressionin WT-Maru, 241AD and 234AD cerebral brain organoids were measured using western blotting technique and quantified using ImageJ. Statistical analysis was performed by Student’s t-test, *P \u0026lt; 0.05, **P \u0026lt; 0.01. The results are presented as the average±SD (n=3, 3 organoids per sample, total of 9 organoids were used). D) Lysates of 241AD and 234AD cerebral brain organoids were analyzed by ELISA at d98. The amount of Aβ1-40 and Aβ1-42 in the RIPA fraction were measured using Aβ42/Aβ40 ELISA KIT (n=3, 3 organoids per sample, total of 9 organoids were used).\u003c/p\u003e","description":"","filename":"GOFigure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/e8b21ad2dd4cea8ff05042e1.jpg"},{"id":104407213,"identity":"6dd8dce8-8d8a-4bf9-a7d9-1ab6c173a676","added_by":"auto","created_at":"2026-03-11 12:35:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5282225,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/93ce8a30-5454-4fd0-97ef-19308ce87657.pdf"},{"id":103338642,"identity":"3c045c00-4e1b-453e-8edd-bd426acdb3f8","added_by":"auto","created_at":"2026-02-24 15:07:02","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":35113,"visible":true,"origin":"","legend":"","description":"","filename":"daNGOParticleSizeAnalysis.csv","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/42bf7d16f2db27c63666cbbb.csv"},{"id":103506909,"identity":"2104f6eb-f46c-4d35-80d1-b3abf1872487","added_by":"auto","created_at":"2026-02-26 13:39:53","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1511978,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSupplementalFigure 1. Brain Organoid Generation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA) Representative image of WT-CMC3, WT-Maru, PSEN1 AD, 241AD and 234AD cerebral brain organoid generation at different stages. Scale bars showing 1mm. B) Average growth of cerebral brain organoids measured in terms of area at different stages. The results are presented as the average±SD (n=10).\u003c/p\u003e","description":"","filename":"GOFigureSupplement1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/71b900b11d676b03f276ba3d.jpg"},{"id":103338632,"identity":"7005c489-dfba-4f0b-9ea1-b19fdae295f8","added_by":"auto","created_at":"2026-02-24 15:07:01","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":605502,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSupplemental Figure 2. NGO Characterization\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA) Particle lateral size measurements shown as a distribution graph, showing average value of 10nm and 99.9% distribution between 5nm to 33.7nm. A total of 2.8x10\u003csup\u003e12\u003c/sup\u003e particles were measured, measured raw data is presented in Table 1. B) Attenuated total reflectance fourier transform infrared spectroscopy (ATF-FT-IR) graph, each arrow and box representing peak wavelength of designated chemical bonds.\u003c/p\u003e","description":"","filename":"GOFigureSupplement2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/2547e06b92f6503545ef917a.jpg"},{"id":103505955,"identity":"ca77122b-6292-4e0b-b158-3c3460ed5331","added_by":"auto","created_at":"2026-02-26 13:33:38","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2428457,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSupplemental Figure 3. No Effect of NGO on Brain Organoid Neurogenesis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA) Representative image of PSEN1 cerebral brain organoids and average area calculation at d84 and d98 for NGO non-treated and treated group. Scale bars showing 1cm. Statistical analysis was performed by Student’s t-test. The results are presented as the average±SD (n=10). B) Representative images of immunostaining of WT and PSEN1 cerebral brain organoids at d98 with or without NGO treatment; neuronal markers (TUJ1, MAP2), neuroepithelium marker (SOX2), proliferation marker (Ki67). Nuclei were stained with DAPI. Scale bars showing 100μm. Statistical analysis was performed by Student’s t-test. The results are presented as the average±SD (n=6). \u0026nbsp;C) Amount of neuronal marker (TUJ1 and NeuN) expressions were visualized using western blotting technique and quantified using ImageJ. Statistical analysis was performed by Student’s t-test. The results are presented as the average±SD (n=3, 3 organoids per sample, total of 9 organoids were used). Unedited versions of all western blot images are shown and labelled in supplementary figure 4.\u003c/p\u003e","description":"","filename":"GOFigureSupplement3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/ea8c962136919747bdbf4a8f.jpg"},{"id":103506363,"identity":"18af3f44-397e-48f7-b5c2-9404eb283c8a","added_by":"auto","created_at":"2026-02-26 13:35:38","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1086337,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSupplemental Figure 4. Full Length Western Blots\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA) Unedited images of western blots in figure 4F.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"GOFigureSupplement4a.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/d88dad39ea09bd64604d6d21.jpg"},{"id":103338645,"identity":"c390d819-8e99-4a0c-839b-1d85320443bb","added_by":"auto","created_at":"2026-02-24 15:07:02","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1293722,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eB) Unedited images of western blots in figure 5a.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"GOFigureSupplement4b.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/595f2f1a334f8eb7924d845d.jpg"},{"id":104397455,"identity":"297dc28e-34dc-432a-9961-c860215a6d1b","added_by":"auto","created_at":"2026-03-11 11:48:40","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1213432,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eC) Unedited images of western blots in figure 5c.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"GOFigureSupplement4c.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/207c593b46f72395d9837e08.jpg"},{"id":103507045,"identity":"524d5607-00a5-4e30-af3c-8b53033744b6","added_by":"auto","created_at":"2026-02-26 13:40:17","extension":"jpg","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":970297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eD) Unedited images of western blots in figure 7d. E) Unedited images of western blots in figure 5c. D) Unedited images of western blots in figure 8c. F) Unedited images of western blots in supplementary figure 3c.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"GOFigureSupplement4d.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8383146/v1/667df6b761491f75b1111b0a.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Autophagy and neuroinflammation modulation by nano-graphene oxide in PSEN1 Alzheimer’s brain organoids","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD) is a neurodegenerative disease affecting more than 50\u0026nbsp;million individuals worldwide, and its research is a rapidly growing field that strives to identify the underlying causes of AD phenotypes, as well as to develop effective drugs or treatments. In terms of familial AD (FAD), patients tend to develop AD symptoms at much younger ages than sporadic AD (SAD) patients [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Autosomal dominant genetic mutations such as amyloid precursor protein (APP) or presenilin-1 (PSEN1) mutations are known to cause the most severe forms of AD [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Researchers have been utilizing a variety of techniques, including genetic studies, brain imaging, and animal models, to learn more about the changes that occur inside the brain as AD progresses [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The major hallmark of AD is now known as hyperphosphorylated tau (pTau)-derived neurofibrillary tangles (NFTs) and the neuronal accumulation and consequent toxicity of amyloid-beta (Aβ) plaques [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These establishments have aided in clinical trials of new ongoing treatments; however, despite extensive progress in the neuroscience of AD, no significant improvements were made in the areas of AD treatments [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the past couple of decades, there have been multiple attempts at manufacturing an \u003cem\u003ein vitro\u003c/em\u003e experimental platform to mimic the structural and functional architectures of the human brain [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, creating a reproducible research platform which could potentially incorporate fully or even partially mimicking the human brain has been very challenging [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In order to overcome such challenges, the use of stem cells and 3D \u003cem\u003ein vitro\u003c/em\u003e modelling systems have been brought up by multiple research groups. Brain organoids are 3D cultures of brain cells derived from induced pluripotent stem cells (iPSCs) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These organoids offer a number of advantages for studying the brain and neurological diseases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. For instance, organoids provide a way to study brain development and the underlying mechanisms of certain neurodegenerative diseases, such as Parkinson\u0026rsquo;s and Alzheimer\u0026rsquo;s diseases, in a controlled and realistic environment [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Moreover, brain organoids provide special platforms where one could elaborate on utilizing viral infections, such as Zika and SARS-CoV-2 infection, and examine different phenotypes caused by specific viral infection [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Even though mouse models are a frequently used research model in neurodegenerative disease research, given the major differences between different species, phenotypes displayed in the human AD brain are not fully represented in the mouse brain AD model [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGraphene, a hexagonal lattice of carbon atoms, displays varied properties influenced by different size and functional groups [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Graphene oxide (GO), a derivative modified with oxygen-containing groups like carboxyl and hydroxyl, and nano-graphene oxide (NGO), with dimensions below 100 nm and depth of only couple of sheets, exhibit unique characteristics over conventional GO; such as quantum confinement and edge effects [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Due to their compatibility with biological systems and ability to modulate immune responses, NGOs have gained traction in biomedical applications [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Despite efforts to incorporate GO into reinforcing biopolymers, still remains a lack of understanding regarding the \u003cem\u003ein vivo\u003c/em\u003e effects of NGOs, particularly in the context of excretion [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. As the therapeutic use of nanoparticles strongly depends on the size of the particles, we have manufactured GO with average lateral size of 10nm and named as nano-graphene oxide (NGO) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe purpose of this study was to assess the effectiveness of NGO on AD cerebral brain organoids produced from patient-derived iPSCs. We hypothesized that NGO could potentially reduce AD phenotypes by activating autophagic mechanisms and diminishing neuroinflammation. Overall, the results suggest that this AD brain organoid model can serve as a useful tool to test treatments for AD patients.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eiPSC Culture\u003c/h2\u003e \u003cp\u003eWe obtained five human-iPSC cell lines for this study. One wildtype iPSCs; CMC-hiPSC-003 (WT-CMC3), and two AD patient-derived iPSCs; UCSD241i-APP2-3 (241AD) and SCD234i-SAD2-3 (234AD), were provided by National Stem Cell Bank of Korea (Korea National Institute of Health, South Korea), and for the PSEN1-mutated patient-derived iPSC line (PSEN1 AD), the CS40iFAD-nxx (RRID:CVCL_YX94) cell line was purchased from Cedar-Sinai Medical Center iPSC Core Facility. Also, another wildtype iPSC; WT-Maru, was provided by Maru Therapeutics (Maru Therapeutics, Seoul, South Korea). Both WT-iPSCs and AD-iPSCs were used to generate wild-type and AD cerebral brain organoids, respectively. WT-CMC3 iPSCs were cultured on vitronectin (Gibco, Massachusetts, USA)-coated plates and cultured using Essential 8\u0026trade; Medium (Gibco, Massachusetts, USA), WT-Maru iPSCs were cultured on GFR-Matrigel (Corning, Massachusetts, USA)-coated plates using STEMFLEX\u0026trade; Medium (Gibco, Massachusetts, USA), PSEN1 iPSCs were cultured on GFR-Matrigel (Corning, Massachusetts, USA)-coated plates and cultured using mTeSR\u003csup\u003eTM\u003c/sup\u003e-Plus medium (STEMCELL Technologies, Canada), 241AD iPSCs were cultured on GFR-Matrigel (Corning, Massachusetts, USA)-coated plates and cultured using mTeSR\u003csup\u003eTM\u003c/sup\u003e-Plus medium (STEMCELL Technologies, Canada) and 234AD iPSCs were cultured vitronectin (Gibco, Massachusetts, USA)-coated plates and cultured using E8-mTeSR\u0026trade; medium (STEMCELL Technologies, Canada) as instructed by the manufacturers. All of the cell lines were passaged using ReLeSR\u0026trade; (STEMCELL Technologies, Canada) when confluent.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBrain Organoid Generation\u003c/h3\u003e\n\u003cp\u003eGeneration of brain organoids was performed following the protocol previously described by Lancaster et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In brief, iPSCs were dissociated into single cells using TryPLE\u0026trade; express enzyme (Thermo Fisher Scientific, Massachusetts, USA). Dissociated iPSCs were then seeded onto ultralow binding 96-well plates (Corning, Massachusetts, USA), with 9,000 cells in each well with mTeSR\u003csup\u003eTM\u003c/sup\u003e-Plus medium, 4 ng/ml basic fibroblast growth factor (Sigma‒Aldrich, Missouri, USA) and 50 \u0026micro;M Rho-associated protein kinase inhibitor. Then, the medium was replaced daily with fresh medium when EBs were formed. Then, as the diameter of EBs reached 500 \u0026micro;m, they were transferred to low-adhesion 24-well plates (Corning, Massachusetts, USA) with neural induction medium consisting of DMEM-F12 (Gibco, Massachusetts, USA) with 1x N-2 Supplement (Gibco, Massachusetts, USA), 1x GlutaMAX (Gibco, Massachusetts, USA), 1x MEM-NEAA (Gibco, Massachusetts, USA) and 1 \u0026micro;g/ml heparin solution. After 6 days, neuroectodermal tissues were embedded in Matrigel droplets (Corning, Massachusetts, USA). These droplets were incubated at 37\u0026deg;C for 20 minutes to solidify and cultured in cerebral organoid differentiation medium without vitamin A (CODM-wo) consisting of a 1:1 ratio of DMEM-F12 (Gibco, Massachusetts, USA) and Neurobasal Medium (Gibco, Massachusetts, USA) with 1x N-2 Supplement (Gibco, Massachusetts, USA), 1x B-27 Supplement without Vitamin A (Gibco, Massachusetts, USA), 1x 2-mercaptoethanol (Sigma-Aldrich, USA), 1x insulin (Sigma-Aldrich, USA), 1x GlutaMAX (Gibco, Massachusetts, USA) and 1x MEM-NEAA (Gibco, Massachusetts, USA) for 4 days. Then, the medium was replaced with CODM composed of a 1:1 ratio of DMEM-F12 (Gibco, Massachusetts, USA) and Neurobasal Medium (Gibco, Massachusetts, USA) with 1x N-2 Supplement (Gibco, Massachusetts, USA), 1x B-27 Supplement (Gibco, Massachusetts, USA), 1x 2-mercaptoethanol (Sigma-Aldrich, USA), 1x insulin (Sigma-Aldrich, USA), 1x GlutaMAX (Gibco, Massachusetts, USA) and 1x MEM-NEAA (Gibco, Massachusetts, USA).\u003c/p\u003e\n\u003ch3\u003eNGO and NGO-biotin Production and Characterization\u003c/h3\u003e\n\u003cp\u003eNGO was produced and provided by INBCT Co., LTD (Seoul, Korea). NGO was synthesized from graphite via Taylor-Couette flow and stabilized solid state of NGO powder was suspended in deionized water. NGO was then loaded onto a 400-mesh carbon coated cooper grid for morphology analysis using Cs-corrected HRTEM (JEM-ARM200F, Cold FEG, JEOL Ltd, Japan). The size distribution of NGO was analyzed using a CPS disc centrifuge (CPS Instruments, USA). Next, NGO was then loaded onto sapphire wafers and then scanned by atomic force microscopy in noncontact mode (scanned area 25 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e, XE-100, Park Systems, Korea). NGO was also provided by INBCT Co., LTD (Seoul, Korea). The method of producing biotin tagged NGO followed similar protocol as producing NGO, with an extra modification and sterilization using high resistance nonspecific probe displacement technique.\u003c/p\u003e\n\u003ch3\u003eNGO and NGO-biotin Treatment\u003c/h3\u003e\n\u003cp\u003eOn Day 84, both WT and AD brain organoids were exposed to NGO or NGO-biotin at a concentration of 10 \u0026micro;g/ml. Organoids were treated for 2 weeks with the CODM\u0026thinsp;+\u0026thinsp;NGO medium or CODM\u0026thinsp;+\u0026thinsp;NGO-biotin medium, changed every other day.\u003c/p\u003e\n\u003ch3\u003eMTT Assay\u003c/h3\u003e\n\u003cp\u003eiNSCs were seeded at 5 x 10\u003csup\u003e4\u003c/sup\u003e cells/well in a 24-well plate in iNSC maintenance medium. After 24 h, cells were treated with A-769662 at different concentrations for 48 h. Then, 500 \u0026micro;l of MTT solution at 500 \u0026micro;g/ml concentration was added to each well and incubated for 4 h at 37\u0026deg;C. Then, 500 \u0026micro;l of DMSO was added to each well to dissolve the formazan crystals. The absorbance value at 570 nm was obtained using an Infinite200 PRO microplate reader (Tecan, Switzerland).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA Extraction and Reverse Transcriptase PCR\u003c/h2\u003e \u003cp\u003eThree cerebral brain organoids in each experimental group were lysed in 1 ml of TRIzol (Invitrogen, Massachusetts, USA) for RNA extraction according to the manufacturer\u0026rsquo;s instructions. cDNA synthesis was performed using Superscript-III First-Strand KIT (Invitrogen, Massachusetts, USA) for RT‒PCR analysis. RT‒PCR was performed with SYBR Green PCR Mix (Applied Biosystems, Massachusetts, USA) using a 7500 Real-Time PCR system (Thermo Fisher Scientific, Massachusetts, USA) carried out with initial denaturation conditions at 50\u0026deg;C for 2 min and at 95\u0026deg;C for 10 min followed by 95\u0026deg;C for 15 s and 60\u0026deg;C for 1 min for at least 60 cycles. All readings were normalized to the level of GAPDH, the housekeeping gene. The list of primers used, and their sequences are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern Blot Analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern Blot Analysis\u003c/div\u003e \u003cp\u003eThree cerebral brain organoids in each experimental group were lysed with 300 \u0026micro;l of Pro-Prep protein lysis buffer (Intron Biotechnology, South Korea) and Sonifier450 (Branson, Connecticut, USA). The concentration of protein samples was measured using a Bio-Rad DC protein assay kit (Bio-Rad Laboratories, California, USA), alongside bovine serum albumin (BSA) as the standard. Each sample was separated using 6%, 10% and 15% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and transferred onto a nitrocellulose membrane. The membrane was then blocked with 3% BSA and incubated overnight with a single primary antibody at 4\u0026deg;C. After another 1 h incubation with the appropriate secondary antibody, the proteins were detected with enhanced chemiluminescence (ECL) reagent (GE Healthcare Life Science, UK) and then viewed using ChemiDocTM MP (Bio-RAD, California, USA)\u003c/p\u003e\n\u003ch3\u003eHistology and Immunofluorescence\u003c/h3\u003e\n\u003cp\u003eOrganoids were fixed in 4% paraformaldehyde for 1h at RT. Each organoid was washed with PBS and incubated in 30% sucrose solution for at least 24h at 4\u0026deg;C. The sample was then embedded in sucrose/gelatin solution and rapidly frozen using liquid nitrogen. Samples were then sectioned into thin slices and used for immunostaining.\u003c/p\u003e \u003cp\u003eSectioned samples were washed and then placed under permeabilization and blocking solution. Each sample was incubated with specific primary antibodies overnight at 4\u0026deg;C. After another 1h incubation with matching secondary antibodies, the samples were then stained with DAPI and mounted using DAKO fluorescence mounting medium (Agilent Pathology Solutions, California, USA).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eThe protein levels of Aβ40 and Aβ42 in RIPA buffer were measured using human Aβ40 ELISA Kit (Thermo Fisher Scientific, Massachusetts, USA) and Aβ42 ELISA Kit (Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturer\u0026rsquo;s instructions and quantified using BCA analysis. RIPA lysates of 3 cerebral brain organoids were used to create a single reading of Aβ40 and Aβ42 and each group was measured in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eADP/ATP Ratio Assay\u003c/h2\u003e \u003cp\u003eThe ADP/ATP ratio assay was performed according to the manufacturer\u0026rsquo;s instructions. In short, 3 cerebral brain organoids per group were dissociated using Accutase and seeded onto 96-well plates at 10\u003csup\u003e4\u003c/sup\u003e cells per well. ATP reagent mix was added to each well and incubated for 1 min at RT. Then, ATP luminescence was recorded, RLU\u003csub\u003eA\u003c/sub\u003e. After another 10 min incubation at RT, ADP luminescence was recorded, RLU\u003csub\u003eB\u003c/sub\u003e. Immediately after the ADP luminescence was recorded, ADP reagent mix was added and incubated for 1 min at RT, and then, luminescence was recorded, RLU\u003csub\u003eC\u003c/sub\u003e. All experiments were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAMP Colorimetric Assay\u003c/h2\u003e \u003cp\u003eThe AMP colorimetric assay was performed according to the manufacturer\u0026rsquo;s instructions. In short, 3 cerebral brain organoids per group were dissociated using AMP assay buffer. After centrifugation at 10,000 g at 4\u0026deg;C for 10 min, the supernatant was collected, and 20 \u0026micro;l was placed onto a 96-well plate with 30 \u0026micro;l of AMP assay buffer. Then, 50 \u0026micro;l of reaction mix was added to each well and incubated for 60 min at 37\u0026deg;C. After incubation, absorbance was recorded at 570 nm. All experiments were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eQUANTSeq mRNA-Sequencing Analysis\u003c/h2\u003e \u003cp\u003emRNA sequencing analysis was performed on 2 samples per group: WT Cont, WT NGO, PSEN1 Cont and PSEN1 NGO. Each RNA sample was created using 3 organoids undergoing total RNA isolation using 1ml of TRIzol (Invitrogen, Massachusetts, USA) according to the manufacturer\u0026rsquo;s instructions. All of the RNA samples\u0026rsquo; quality control was done using Bioanalyzer 2100 system using ND-2000 spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA). Each sample underwent cDNA library construction and sequencing using a QuantSeq 3\u0026prime; mRNA-Seq Library Prep Kit (Lexogen Inc., Austraia), following the manufacturer\u0026rsquo;s protocol. High-throughput sequencing was conducted as single-end 75, generating approximately 10\u0026nbsp;million reads per sample, utilizing a NextSeq 500 (Illumina Inc., California, USA). For further analysis, ExDEGA (eBiogen, South Korea) gene analysis tool was used to determine differentially expressed genes with fold change equal or higher than 2 and p-value of equal or less than 0.05. The clustering heatmaps and gene ontology data were created using SRplot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData analyses in all related experiments were carried out with Prism 9 software (GraphPad Software, California, USA), and statistical analyses were performed depending on the number of groups, distribution, variance, and normality. Two-tailed Student\u0026rsquo;s t test analysis and one-way ANOVA were employed for parametric datasets, and the level of significance is shown as *** P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ** P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, or * P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or ns for not significant. The number of biological replications performed in different experiments is shown in each figure legend. Furthermore, all samples were chosen randomly for analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGeneration and characterization of WT and PSEN1 patient-derived cerebral brain organoids\u003c/h2\u003e \u003cp\u003eIn this study, two iPSC lines were employed to simulate AD. CMC-hiPSC-003 cells were utilized as the WT-CMC3 control group, while PSEN1 iPSCs were used as the experimental group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These cell lines were used to generate cerebral brain organoids using a protocol that was previously established with slight modifications [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Briefly, approximately 9000 iPSCs were seeded onto a single ultralow-attachment 96-well plate for 6 days to form an embryoid body. On Day 6, EBs were collected and underwent neural induction for 4 days. On Day 11, each organoid was placed inside a Matrigel droplet placed in CODM medium without vitamin A for neuroepithelial bud expansion and then transferred to CODM medium with vitamin A for further expansion. Both WT-CMC3 and PSEN1 AD organoids were cultured for up to 12 to 14 weeks of maturation and then sampled at different time points for further analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The size of the brain organoids from both WT-CMC3 and PSEN1 mice showed fairly even growth. The PSEN1 AD brain organoids were slightly smaller than the WT-CMC3 organoids on Day 56; however, both groups showed similar sizes on Day 84 (Supple Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The average area of the organoids on Day 42 was determined, and sections of the WT-CMC3 and PSEN1 AD brain organoids both showed clear expression of the cell proliferation marker KI-67, neuronal progenitor marker SOX2, and neuronal markers TUJ1 and MAP2. These markers were also expressed on Day 84 in both the WT-CMC3 and PSEN1 AD brain organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These immunostaining images illustrate that brain organoids generated from PSEN1-mutated patient-derived iPSCs exhibited similar levels of brain development compared to WT-CMC3 brain organoids. The results of quantifying specific markers through ImageJ software also showed that the development and maturity of the PSEN1 AD brain organoids were comparable to those of the WT brain organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Collectively, both the WT-CMC3 and PSEN1 AD brain organoids effectively mimicked neurodevelopment in organoid cultures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePSEN1 AD brain organoids were generated using iPSCs derived from an AD patient with the PSEN1 A246E mutation. This mutation is known to have altered catalytic site conformation on γ-secretase, which results in an impaired 4th cleavage site. This alteration affects the cleavage site of APP, which then leads to an increase in the ratio of Aβ42/Aβ40 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). With this genetic alteration, PSEN1 AD brain organoids are expected to show an increase in AD phenotypes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Tau immunostaining was observed in both the WT-CMC3 and PSEN1 AD brain organoids at all time points. In the case of the AD phenotypic markers pTau and Aβ, neither marker was expressed in the WT-CMC3 brain organoids at either Day 42 or Day 84. In the PSEN1 AD brain organoid samples, pTau and Aβ were expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The quantified expression of AD phenotypic markers illustrate that Aβ tend to increase at a faster rate when cultured for a longer period of time. On the other hand, the growth of pTau expression tends to occur but at much slower rate than Aβ (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eNano-graphene oxide characterization\u003c/h2\u003e \u003cp\u003eIn order to categorize NGO used in this study as nanoparticles, particle size and the distribution were measured. Atomic force microscopy (AFM) was used to measure the thickness of NGO. AFM measurement showed that the thickness of NGO is around 0.72nm and 0.77nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Since the thickness of a single sheet layer of graphene oxide is to be below 1.2nm, AFM shows that NGO is composed of single layer NGOs [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Transmission electron microscopy (TEM) method was then used to measure the size of the NGO. TEM image illustrates that most of the NGO particles are smaller than 20nm scale bar used in the image (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). We also measured about 2.8x10\u003csup\u003e12\u003c/sup\u003e particles in total and created a lateral size distribution graph which states the average size of each NGO particle is around 10nm and that 99% of the particles are distributed within the range of 5nm to 33.7nm (Supple Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFourier transform infrared spectroscopy (FT-IR) was measured using Film and KBr pellet method. Chemical bond groups present within NGO are visualized at relative wavelengths; O-H at 3426cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, C-H at 2920cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2853cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, C\u0026thinsp;=\u0026thinsp;O at 1731cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, C\u0026thinsp;=\u0026thinsp;C at 1629cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, C-OH at 1387cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and C-O at 1025cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Attenuated total reflectance fourier transform infrared spectroscopy (ATF-FT-IR) was also measured. C\u0026thinsp;=\u0026thinsp;O, -OH bending vibration and C\u0026thinsp;=\u0026thinsp;O were shown in 1733cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1635cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1630cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e wavenumber, respectively and carbon single bonded hydrogen bending, and carbon single bonded oxygen stretching were located and marked at their established positions (Supple Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Criteria for a compound to be labelled as nanoparticle, physical properties, such as size within the range of 1 to 100 nm and significantly larger surface area to volume ratio, and chemical properties, such as surface chemistry, must be met [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Both physical and chemical properties shown above using TEM, FT-IR and ATF-FT-IR clearly dictate NGO as nanomaterial.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEffect of NGO treatment on PSEN1 AD cerebral organoids\u003c/h2\u003e \u003cp\u003eAs shown above, phenotypic markers of AD are expressed at a higher level at Day 84; therefore, we added NGO to the PSEN1 AD brain organoids at Day 84 for a 2-week period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). NGO was administered at a concentration of 10 \u0026micro;g/ml, and when treated for a 2-week period, the organoids seemed to be covered by NGO (Supple Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To prove that NGO successfully penetrated into the organoid, we used biotin-tagged NGO, and immunostaining images of biotin confirmed that NGO could passively diffuse into the brain organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The NGO-treated group also did not show any cytotoxic phenotypes compared to the nontreated group. The level of c-cas3 expression did not differ in all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), and the area of the NGO-treated organoids did not differ compared to that of the nontreated organoids at Day 98 (Supple Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In terms of neural development, immunostaining images of TUJ1, SOX2, MAP2 and KI67 showed no difference between the treated and nontreated groups within WT-CMC3 and PSEN1 AD brain organoids (Supple Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Furthermore, this finding was supported by western blot images and quantified levels of Tuj1 and NeuN protein expression, as there were no significant differences between the treated and nontreated groups within WT-CMC3 and PSEN1 AD brain organoids (Supple Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In summary, NGO treatment did not cause any cytotoxic activity or affect the development of brain organoids.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs expected, there were changes in the expression level of AD phenotypes. In the case of pTau expression, both the treated and nontreated groups of PSEN1 AD brain organoids showed pTau expression. However, the level of Aβ secretion significantly decreased in the NGO-treated group compared to the nontreated group. This result was evident in both immunostaining images and quantified Aβ expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). ELISA data also showed that the ratio of Aβ42/Aβ40 in the NGO-treated group decreased compared to that in the nontreated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Furthermore, western blot analysis of Aβ showed decreased expression of Aβ in the NGO-treated group compared to the nontreated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These data confirm that NGO can decrease Aβ secretion levels in PSEN1 AD brain organoids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eNGO reduces Aβ levels via an autophagic mechanism\u003c/h2\u003e \u003cp\u003eTo further investigate NGO\u0026rsquo;s mechanism of action on AD brain organoids, we examined multiple mechanisms by which NGO decreased Aβ levels. Li et al. have shown that graphene oxide could have an effect on inducing autophagy on a 2D cellular level [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] Therefore, we wanted to check if our NGO could also induce autophagy on human cerebral organoid. We screened for a list of proteins related to autophagy using western blotting analysis and found an increase in the level of Beclin1 in the NGO-treated group. Furthermore, in combination with the decrease in the level of p62 in the NGO-treated group, NGO treatment at a concentration of 10 \u0026micro;g/ml for a 2-week period activated the autophagic mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, we examined the expression levels of LC3b, mTOR and AMPK. As we examined the phosphorylated forms of both mTOR and AMPK, NGO treatment elevated the ratio of p-AMPK expression to AMPK expression and decreased the ratio of p-mTOR to mTOR expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). These outcomes indicate that NGO treatment inhibits mTOR activation by activating the AMPK signaling pathway in PSEN1 AD brain organoids. AMPK can be activated by two mechanisms: direct activation via an increase in ROS levels by modifying the AMPKα subunit and indirect activation via an increase in AMP and ADP levels [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. To examine the detailed mechanism of NGO, we examined AMP, ADP and ATP levels in the NGO-treated and untreated PSEN1 AD brain organoids. As positive control, a potent AMPK indirect activator, A-769962, was employed. 2D induced neural stem cells (iNSCs) have been utilized in finding the optimal concentration of A-769962. iNSC is a reprogrammed cell line obtained from human fibroblast, as described in our previous study [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The optimal concentration was shown to be 100 \u0026micro;M after a cytotoxicity assay performed on 2D iNSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). In order to measure the effectiveness of NGO activating AMPK pathway, AMP/ATP ratio is required. This value was obtained using two different analysis tools: ADP/ATP ratio assay kit and AMP assay kit. Then by utilizing adenylate kinase equilibrium equation, AMP/ATP ratio was calculated. The NGO-treated group showed an increased AMP/ATP ratio compared to the control group; however, this ratio was slightly lower than that of A-769962 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Activation of AMPK by NGO was also verified by measuring sirtuin 2 (SIRT2) level and ATG7. SIRT2 is known to enhance AMPK activation by deacetylating LKB1, which in turn promotes ATG7-mediated autophagosome formation. Both genes were upregulated in NGO treated group, which indicates an increased level of both SIRT2 and ATG7 promotes AMPK activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eGene expression analysis of WT and PSEN1 AD cerebral organoids\u003c/h2\u003e \u003cp\u003eAs mentioned above, the phenotypical hallmark of AD is well known to be the aggregation of Aβ [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Even though treatment of NGO increases autophagy activity within the organoid, which then leads to removal of aggregated Aβ, however, this alone does not provide clear explanation on decrease in Aβ. Therefore, we utilized whole-transcriptome RNA sequencing to screen and identify gene expression differences between WT and PSEN1 AD cerebral organoids and also to discover any other therapeutic effect of NGO treatment AD cerebral organoids. RNA samples: WT-CMC3, NGO treated WT-CMC3, PSEN1 AD and NGO treated PSEN1 AD organoids at d98, were used to identify genes that were differentially expressed between different groups with or without NGO treatment. After 14 weeks of culture of 4 groups, diversifications were observed between all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). mRNA sequencing of four groups resulted in identifying around 43,000 genes, of which there were around 8,500 genes that showed statistically significant differences between groups. From the list of statistically significant differential gene expressions, the top 550 genes were selected for gene ontology analysis, including biological processes (BP), cellular components (CC), and molecular function (MF) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Enrichment scores in all three ontologies illustrated an increase in multiple areas; but especially several sections were highly correlated with neural inflammation. For further simplification of illustrated gene ontology results, data were then categorized and linked with related genes and then visualized using Cnet plot of enriched KEGG pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) and then transformed into a bar graph (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). KEGG pathway analysis discovered significant enrichments in areas correlated with inflammatory cytokines and their mechanisms, such as, cytokine-cytokine receptor interaction, TLR signaling pathway and NF-kappa B signaling pathway. Pathway analysis also indicated differential gene expression also linked with IL-17 pathway, which could implicate the relationship on NGO working within IL-17 signaling cascade. RNA-sequencing analysis once again illustrated the relationship between NGO treatment and neural inflammation, which then allowed us to further investigate the detailed mechanism of NGO on Aβ reduction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eNGO reduces the level of newly formed Aβ by reducing proinflammatory cytokines\u003c/h2\u003e \u003cp\u003eAs stated above, NGO activates autophagy by activating the AMPK signaling pathway, which then leads to the removal of secreted Aβ in PSEN1 AD brain organoids. Then, we examined whether NGO negatively participates in the formation of Aβ. NGO is known to have both proinflammatory and anti-inflammatory effects depending on the concentration and type of NGO [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, KEGG pathway analysis also illustrated that inflammation related pathways were enriched; thus, we examined how NGO affects inflammation within AD brain organoids. To validate the inflammatory response of NGO on PSEN1 AD brain organoids, we examined the expression levels of both pro- and anti-inflammatory cytokines via RT‒PCR. In a comparison of the NGO-treated group and the nontreated group, there was a decrease in the expression of the proinflammatory cytokines TNF and IL-1A, whereas there was no difference between the groups in the anti-inflammatory cytokines IL-10 and TGFβ (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The presence of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (IBA1) expression in both groups indicates that astrocytes and microglial cells are expressed in cerebral brain organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Microglial cells are known to express inducible nitric oxide synthase (iNOS) when in response to inflammatory stimuli. However, the decrease in pro-inflammatory cytokines, such as TNF and IL-1A, due to NGO treatment, lowers the activation of microglia and astrocytes, which then leads to lower iNOS expression. Also, since TNF acts on microglia and astrocytes to induce C3, lowered expression of TNF leads to lower expression of C3 in NGO treated group compared than control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Due to the decrease in proinflammatory cytokine levels and decrease in the activation of astrocytes and microglial cells, it has been shown that the level of interferon-induced transmembrane protein 3 (IFITM3) expression also decreased in the NGO-treated group by immunostaining (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). A decrease in IFITM3 levels also supported western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Also, as shown in the RNA sequencing data, we looked further into the NFkB signaling pathway. The decrease in the activation of NFkB alongside stable ERK1/2 expression but decrease in COX2 and IL-1b expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD) illustrates that NGO inhibits NFkB activity by increasing the expression of NFkb inhibitor beta (NFkBIB). In summary, NGO treatment decreased Aβ levels in PSEN1 AD brain organoids by decreasing NFkBIB and proinflammatory cytokines, which then led to inhibition of γ-secretase activity via a decrease in IFITM3 expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eValidation of NGO treatment on different patient-derived AD brain organoids\u003c/h2\u003e \u003cp\u003eTo examine whether NGO could be a new therapeutical agent in treating AD or it is only suitable for PSEN1 A246E mutated patients, we obtained three more iPSCs; one WT iPSC cell line and two AD patient-derived iPSCs, for NGO validation. WT-Maru was obtained from Maru Therapeutics, wild-type iPSC that has different culturing conditions compared to WT-CMC3. Also, for AD patient-derived iPSCs, UCSD241i-APP2-3 (241AD) and CSD234i-SAD2-3 (234AD) were obtained from National Stem Cell Bank of Korea. 241AD iPSC is from a familial Alzheimer\u0026rsquo;s disease patient, similar to PSEN1 AD iPSCs, with an APP duplication genetical mutation and 234AD iPSC is from a sporadic Alzheimer\u0026rsquo;s disease patient diagnosed at the age of 78. Cerebral brain organoids were generated using the same protocol as above and each group; WT-Maru, 241AD and 234AD, were sampled at d42, d84 and d98 for analysis. These organoids were treated using the same NGO at identical concentration, used with WT-CMC3 and PSEN1 AD brain organoids. Sampled organoids at d42 and d84 were examined via immunohistochemistry, illustrating cell proliferation marker KI-67, neuronal progenitor marker SOX2, and neuronal markers TUJ1 and MAP2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Organoids from all three groups showed clear rosette formation along with clear neuronal development. As these cerebral brain organoids exhibited neuronal development, we then examined for the hallmarks of AD. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, WT-Maru did not express Aβ nor p-Tau. On the other hand, 241AD and 234AD both showed formation of Aβ and p-Tau expression. WT-Maru, 241AD and 234AD brain organoids were also cultured for 12 weeks and then followed by 2 weeks treatment with NGO. Similar to WT-CMC3 and PSEN1 AD brain organoids, the neuronal markers, such as NeuN and Tuj1 expressions, were not affected in all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). In terms of Aβ expression, both 241AD and 234AD showed decrease in Aβ expression, as well as in Aβ-42 over Aβ-40 ratio, there is an obvious decrease in both AD patient-derived brain organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC,D). However, the mechanism of action behind the decreased expression was slightly different. Western blotting images clearly illustrates the obvious increase of beclin1 expression followed by decreased expression of p62 in NGO treated groups, as well as decrease in the ratio of LC3BII to LC3BI clearly indicates that NGO facilitates autophagy mechanism and aid in removing aggregated Aβ. In case of 241AD brain organoids, AMPK activation mechanism of action is very similar to PSEN1 AD brain organoids, as the ratio of p-AMPK/AMPK is significantly higher in NGO treated group than control group in 241AD. However, even though there is a slight increase of p-AMPK/AMPK ratio in 234AD, the statistical analysis shows that the difference is not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). This similar outcome is also shown in inflammatory response mechanism. Starting with decreased expression of COX2 and IL-1b in NGO treated group is similar to PSEN1 AD brain organoid for both 241AD and 234AD organoids. However, even if ERK1/2 level is not changed, the activation of NFkB only occurred in 241AD brain organoids and not in 234AD brain organoids. Similarly, the difference is also found in the IFITM3 expression level. The protein expression level graph illustrates the change between control and NGO treated group in 234AD, but the change is not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAD is a neurodegenerative disorder characterized by progressive cognitive decline and memory loss [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. One of the defining phenotypes of AD is the formation and accumulation of Aβ in the brain. The build-up of Aβ is accompanied by the development of neurofibrillary tangles, which result from the phosphorylation of the Tau protein within nerve fibers. The presence of these neurofibrillary tangles and Aβ deposits leads to the degeneration and loss of neurons in the brain and exacerbates other factors associated with AD, such as inflammation, oxidative stress, and neural microvascular disease [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Despite the major impact of AD on cognitive function, there are currently no treatments that have been proven to be both safe and effective [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This issue is partly due to the limitations of existing animal models and 2D cell culture systems, which do not fully replicate the specific symptoms and indications observed in patients with AD [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The expression of multiple familial Alzheimer's disease (FAD) mutations in animal models accelerates pathological markers that resemble human symptoms. However, despite promising therapeutic effects observed in these models, no treatments have yet demonstrated significant breakthroughs in human clinical trials [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Drummond et al. further highlights that the low success rate\u0026mdash;reported to be as high as 99.6% failure\u0026mdash;in Alzheimer\u0026rsquo;s disease clinical trials underscores the premature translation of pathology reduction in transgenic mice to human applications. To address these limitations, our research aims to confirm that a human cerebral brain organoid model generated from AD patient-derived iPSCs can accurately reproduce the hallmark features of AD. This model can then be used as a platform for testing the efficacy of potential drugs for AD treatment. This approach builds on previous research that has used 3D organoids to model disease in multiple organs, including the liver, brain, and intestine, and the use of brain organoids to study other neurodegenerative diseases such as Parkinson's disease, microcephaly, and amyotrophic lateral sclerosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs shown above, we successfully generated AD cerebral brain organoids using patient-derived iPSCs. After culturing these organoids for an extended period of time, they accurately mimicked both hallmarks of AD phenotypes. Thus, we were able to examine the impact of NGO on AD cerebral brain organoids utilizing this successful high-throughput drug testing platform. Biotin-tagged NGO showed that the size of NGO was an advantage, as it could passively penetrate into the brain organoids without any external force applied. The application of NGO did not physically damage the organoids or cause any neurodegenerative effects. However, when applied at a specific concentration, NGO exerted its therapeutic effects and decreased the level of Aβ by activating multiple signaling pathways.\u003c/p\u003e \u003cp\u003eThe presence of astrocytes and microglia in this cerebral brain organoid model was the key to investigating the inflammatory effect of NGO. Generally, neuroinflammation occurs from microglial activation from increase in proinflammatory cytokines. Proinflammatory cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), bind to receptors in cells and activate signaling pathways that lead to the transcriptional activation of the iNOS gene. Leading to upregulation of C3 and iNOS, cause oxidative stress, mitochondrial dysfunction and potential for neurodegeneration [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In response to the decrease in proinflammatory cytokine levels by NGO treatment, the activation of these signaling pathways will also decrease, leading to a decrease in C3 and iNOS expression and production of nitric oxide (NO). This process can contribute to a reduction in the levels of inflammation in the tissue. These factors had an impact on the reduced IFITM3 secretion in the group treated with NGO. Hur et al. stated that IFITM3 is a γ-secretase modulatory protein that increases inflammatory cytokines and stimulates the expression of IFITM3 in astrocytes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In the NGO-treated group, this effect led to diminished activity of γ-secretase, thus decreasing Aβ formation.\u003c/p\u003e \u003cp\u003eThe activation of AMPK increases autophagy, the process of cellular degradation and recycling [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This activation supports the formation of autophagosomes and enhances lysosomal degradation. This process is believed to maintain cellular stability and protect cells from stress factors. In contrast, mTOR, a regulator of cellular metabolism and growth, obstructs autophagy by blocking autophagosome formation, but AMPK can deactivate mTOR to increase autophagy [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. An increase in beclin1 is a critical component of the autophagic initiation complex and helps promote autophagosome formation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A reduction in the levels of p62, which acts as a scaffold protein in autophagic regulation, releases autophagy-related proteins and activates autophagy. A decrease in the LC3B II/LC3B I ratio is a sign of increased autophagy, as LC3B is involved in autophagic regulation. In terms of AMPK activation by NGO, the exact mechanism by which NGO increases the levels of AMP and ADP is not well understood. However, it is likely that NGO affects cellular energy metabolism, which can lead to changes in the levels of AMP and ADP [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In general, AMP and ADP levels increase when cellular energy stores are depleted, and this increase is sensed by AMPK, which is activated in response [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. NGO may affect cellular energy metabolism through oxidative stress or inflammation, which can lead to changes in the levels of AMP and ADP [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, more research is needed to fully understand the exact mechanism by which NGO affects AMP and ADP levels.\u003c/p\u003e \u003cp\u003eThe effect of NGO treatment was further evaluated using two more AD patient-derived iPSCs; CSD234i-SAD2-3 (234AD) and USCD241i-APP2-3 (241AD). 234AD iPSC is from a sporadic AD patient, who has been diagnosed with AD at the age of 78 and cells were collected at the age of 83, and 241AD iPSC is from a familial AD patient with APP duplication genetic alteration [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Treatment of NGO onto different AD brain organoids also showed similar growth compared to WT brain organoids without any neurodegenerative signals. However, the effect of NGO on each type of AD were both similar and different in some areas. As illustrated above, both 241AD and 234AD showed decrease in Aβ levels, showing promising effect of NGO. However, even though there were slight decrease in the ratio of p-AMPK/AMPK and p-NFkB/NFkB, the level of drop was not shown to be significant. 241AD showed similar effect as PSEN1 AD brain organoids as they are both from Familial Alzheimer's disease (FAD) patients, however, 234AD from sporadic Alzheimer's disease (SAD) patient did not. FAD and SAD exhibit similar underlying pathology and disease progression. However, they differ in genetic etiology and age of onset. FAD is inherited in an autosomal dominant manner and typically manifests earlier in life, whereas SAD, the more prevalent form, has a later onset and results from a complex interaction of genetic predisposition and environmental factors [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Although NGO exhibits therapeutic effects by reducing Aβ levels through modulation of both autophagy and neuroinflammatory pathways, the variability in genetic and environmental factors in SAD patient likely contributes to the observed differences in treatment outcomes [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. To better understand the influence of individual patient-specific factors on therapeutic efficacy, it is essential to conduct systematic screenings utilizing a series of iPSC lines derived from both FAD and SAD patients.\u003c/p\u003e \u003cp\u003eEven though this study specifically illustrates the therapeutical actions of NGO towards AD brain organoid, it also enlightens some limitations that should be addressed. First, due to the fundamental design of an organoid culture system, this model does not have an excretion mechanism. Therefore, the long-term effect of NGOs within the system needs to be studied. Furthermore, even though organoid models show a higher level of similarity to patients than \u003cem\u003ein vivo\u003c/em\u003e models, the effect of NGO treatments on an AD mouse model is needed. Since brain organoid model is not composed with blood-brain barrier system, \u003cem\u003ein vivo\u003c/em\u003e models would be an option in exploring the excretion system as well as whether NGO could pass through the blood‒brain barrier and shows a similar effect compared to an organoid model.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn conclusion, this study suggests the therapeutical applications of NGO towards AD treatment by decreasing aggregation and generation of Aβ. Treatment of NGO on AD patient-derived brain organoid operating via multiple pathways, such as increasing neural autophagic activities by AMPK activation and reducing proinflammatory cytokines and IFITM3 expression via NFkB pathway, strongly illustrate the possibility in utilizing nano graphene oxides for treatment of neurodegenerative diseases.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"330\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eAlzheimer\u0026rsquo;s disease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eAmyloid precursor protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eAPP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eAmyloid-beta\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eA\u0026beta;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eAtomic force microscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eAFM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eAttenuated total reflectance fourier transform infrared spectroscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eAFT-TF-IR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eBiological processes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eBP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eBovine serum albumin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eBSA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eCellular components\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eCC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eEnhanced chemiluminescence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eECL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eFamilial AD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eFAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eFourier transform infrared spectroscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eFT-IR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eGlial fibrillary acidic protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eGFAP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eHyperphosphorylated tau\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003epTau\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eInduced neural stem cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eiNSCs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eInduced pluripotent stem cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eiPSCs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eInducible nitric oxide synthase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eiNOS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eInterferon-induced transmembrane protein 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eIFITM3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eInterleukin-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eIL-1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eInterleukin-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eIL-6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eIonized calcium-binding adapter molecule 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eIBA1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eMolecular function\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eMF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eNano-graphene oxide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eNGO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eNeurofibrillary tangles\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eNFTs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eNFkb inhibitor beta\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eNFkBIB\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eNitric oxide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003ePresenilin-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003ePSEN1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eSporadic AD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eSAD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eTransmission electron microscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eTEM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 74.2424%;\"\u003e\n \u003cp\u003eTumor necrosis factor-alpha\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.7576%;\"\u003e\n \u003cp\u003eTNF-\u0026alpha;\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\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn terms of the iPSC lines used in this study, all iPS cell lines underwent ethics approval under the project title of \u0026ldquo;Therapeutic effect of deca nano-graphene oxide treatment on Alzheimer\u0026rsquo;s disease in the patient-derived Alzheimer\u0026rsquo;s disease brain organoids\u0026rdquo; by Seoul National University Institutional Bioethics Committee, \u003cem\u003eIRB No. 2511/004-016\u003c/em\u003e, 24\u003csup\u003eth\u0026nbsp;\u003c/sup\u003eJune 2024. CS40iFAD-nxx (RRID:CVCL_YX94) cell line is available for purchase for both commercial and academic purposes at https://biomanufacturing.cedars-sinai.org/product/cs40ifad-nxx/. WT-Maru cell line can be provided upon request at https://marurx.com/contact-us/. Ethical approval of WT-Maru can be found at Public IRB number P01-202110-31-009 and published paper with related cells [37]. CMC-hiPSC-003 cell line can be provided upon request at https://nih.go.kr/ncsr/nscb/kr/cdc/scb/scbSalesInfo.do. Ethical approval of CMC-hiPSC-003 can be found at Rim et al. [38] and also at https://nih.go.kr/ncsr. Ethics approval of CSD234i-SAD2-3 and UCSD241i-APP2-3 can be found at Israel et al. [35].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the conclusions of the paper are available in the article and corresponding figures. The sequencing data supporting the results reported in this study have been deposited in the NCBI GEO under the accession number GSE266055. Additional experimental details and more detailed data used or analyzed in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Bio\u0026amp;Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. RS-2023-00266110). This study was partially supported by the Research Institute for Veterinary Science, Seoul National University. This study was partially supported by the Research Institute for Veterinary Science, Seoul National University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e experiments were performed by NGK. Partial iPSC cell lines have been provided by HP and HK. RNA, protein and organoid sampling were performed by NGK and JC. Data analysis was performed by NGK and JC. Data presentation was performed by NGK. RNA-seq was performed by NGK. NGO analysis was performed by NGK, SWC and JCR. Experimental design was performed by NGK and KSK. The manuscript was written by NGK and KSK.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have not used AI-generated work in this manuscript. [김2]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFIRST AUTHOR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNam Gyo Kim\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCORRESPONDING AUTHOR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKyung-Sun Kang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS AND AFFILIATIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNam Gyo Kim, Jaeyong Chun,\u003c/em\u003e \u003cem\u003eHyeyeon Park\u003c/em\u003e, \u003cem\u003eKyung-Sun Kang\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDivision of Intractable Disease Research, National Institute of Health, Osong, Cheongju, 28160, Republic of Korea\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHyeyeon Park, Hyunyoung Kim\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eInstitutes of Convergence Technology, INBCT Co.,LTD, Hwaseong-si 18462, Republic of Korea\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSoon Won Choi, Jae-Chul Ryu\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBreijyeh Z, Karaman R. Comprehensive review on Alzheimer\u0026rsquo;s disease: causes and treatment. Molecules. 2020 Dec 8;25(24):5789.\u003c/li\u003e\n\u003cli\u003eLanoisel\u0026eacute;e HM, Nicolas G, Wallon D, Rovelet-Lecrux A, Lacour M, Rousseau S. APP, PSEN1, and PSEN2 mutations in early-onset Alzheimer disease: A genetic screening study of familial and sporadic cases. PLoS medicine. 2017 Mar 28;14(3):e1002270.\u003c/li\u003e\n\u003cli\u003eChen ZY, Zhang Y. Animal models of Alzheimer\u0026rsquo;s disease: Applications, evaluation, and perspectives. Zoological research. 2022 Nov 11;43(6):1026.\u003c/li\u003e\n\u003cli\u003eRajmohan R, Reddy PH. Amyloid-beta and phosphorylated tau accumulations cause abnormalities at synapses of Alzheimer\u0026rsquo;s disease neurons. Journal of Alzheimer\u0026apos;s Disease. 2017 Jan 1;57(4):975-99.\u003c/li\u003e\n\u003cli\u003eHane FT, Robinson M, Lee BY, Bai O, Leonenko Z, Albert MS. Recent progress in Alzheimer\u0026rsquo;s disease research, part 3: diagnosis and treatment. Journal of Alzheimer\u0026apos;s disease. 2017 Jan 1;57(3):645-65.\u003c/li\u003e\n\u003cli\u003eLovett ML, Nieland TJ, Dingle YT, Kaplan DL. Innovations in 3D tissue models of human brain physiology and diseases. Advanced functional materials. 2020 Oct;30(44):1909146.\u003c/li\u003e\n\u003cli\u003eKim J, Koo BK, Knoblich JA. \u0026quot;Human organoids: model systems for human biology and medicine.\u0026quot; Nature Reviews Molecular Cell Biology 21.10 (2020): 571-584.\u003c/li\u003e\n\u003cli\u003eLee SE, Choi H, Shin N, Kong D, Kim NG, Kim HY. Zika virus infection accelerates Alzheimer\u0026rsquo;s disease phenotypes in brain organoids. Cell Death Discovery. 2022 Apr 2;8(1):153.\u003c/li\u003e\n\u003cli\u003eKook MG, Lee SE, Shin N, Kong D, Kim DH, Kim MS. Generation of cortical brain organoid with vascularization by assembling with vascular spheroid. International Journal of Stem Cells. 2022;15(1):85.\u003c/li\u003e\n\u003cli\u003eLee SE, Kwon D, Shin N, Kong D, Kim NG, Kim HY. Accumulation of APP-CTF induces mitophagy dysfunction in the iNSCs model of Alzheimer\u0026rsquo;s disease. Cell Death Discovery. 2022 Jan 10;8(1):1.\u003c/li\u003e\n\u003cli\u003eKong D, Park KH, Kim DH, Kim NG, Lee SE, Shin N, Kook MG, Kim YB, Kang KS. Cortical-blood vessel assembloids exhibit Alzheimer\u0026rsquo;s disease phenotypes by activating glia after SARS-CoV-2 infection. Cell Death Discovery. 2023 Jan; 9(32). \u003c/li\u003e\n\u003cli\u003eDawson TM, Golde TE, Lagier-Tourenne C. Animal models of neurodegenerative diseases. Nature neuroscience. 2018 Oct;21(10):1370-9.\u003c/li\u003e\n\u003cli\u003eWhitby RL. Chemical control of graphene architecture: tailoring shape and properties. ACS nano. 2014 Oct 28;8(10):9733-54.\u003c/li\u003e\n\u003cli\u003eKim HY, Yoon HH, Seong H, Seo DK, Choi SW, Ryu J. Preventive effects of nano-graphene oxide against Parkinson\u0026rsquo;s disease via reactive oxygen species scavenging and anti-inflammation. BMB reports. 2023 Mar 3;56(3):202.\u003c/li\u003e\n\u003cli\u003eYu A, Park HS, Chae DH, Park JH, Heo J, Cho K, Kim J, Lee H, Jee S, Kim C, Choi SW, Ryu J, Hur EH, Choi Y, Choi EJ, Oh MK, Lee HY, Lee JH, Yu KR. Nanographene Oxide Attenuates Acute GVHD by Modulating Macrophage Polarization in a Xenogeneic Mouse Model. Advanced Science. 2025 Dec 4;12(45). \u003c/li\u003e\n\u003cli\u003eKim DH, Kim MJ, Kwak SY, Jeong J, Choi D, Choi SW. Bioengineered liver crosslinked with nano-graphene oxide enables efficient liver regeneration via MMP suppression and immunomodulation. Nature Communications. 2023 Feb 13;14(1):801.\u003c/li\u003e\n\u003cli\u003eLancaster MA, Knoblich JA. Generation of cerebral organoids from human pluripotent stem cells. Nature protocols. 2014 Oct;9(10):2329-40.\u003c/li\u003e\n\u003cli\u003eEID, May M. Characterization of Nanoparticles by FTIR and FTIR-Microscopy. In: Handbook of consumer nanoproducts. Springer Singapore. 2022; p. 1-30.\u003c/li\u003e\n\u003cli\u003eLi X, Li K, Chu F, Huang J, Yang Z. Graphene oxide enhances \u0026beta;-amyloid clearance by inducing autophagy of microglia and neurons. Chemico-Biological Interactions. 2020 Jul 1;325:109126.\u003c/li\u003e\n\u003cli\u003eKim J, Yang G, Kim Y, Kim J, Ha J. AMPK activators: mechanisms of action and physiological activities. Experimental \u0026amp; molecular medicine. 2016 Apr;48(4):e224-.\u003c/li\u003e\n\u003cli\u003eBałaban J, Wierzbicki M, Zielińska-G\u0026oacute;rska M, Sosnowska M, Daniluk K, Jaworski S. Graphene Oxide Decreases Pro-Inflammatory Proteins Production in Skeletal Muscle Cells Exposed to SARS-CoV-2 Spike Protein. Nanotechnology, Science and Applications. 2023 Dec 31:1-8.\u003c/li\u003e\n\u003cli\u003eJicha GA, Carr SA. Conceptual evolution in Alzheimer\u0026apos;s disease: implications for understanding the clinical phenotype of progressive neurodegenerative disease. Journal of Alzheimer\u0026apos;s Disease. 2010 Jan 1;19(1):253-72.\u003c/li\u003e\n\u003cli\u003eDrummond E, Wisniewski T. Alzheimer\u0026rsquo;s disase: experimental models and reality. Acta Neuropathol. 2016 Dec 26;133(2):155-175.\u003c/li\u003e\n\u003cli\u003eZhang W, Xiao D, Mao Q, Xia H. Role of neuroinflammation in neurodegeneration development. Signal Tansduction and Targeted Therapy. 2023 Jul 12;267.\u003c/li\u003e\n\u003cli\u003eHur JY, Frost GR, Wu X, Crump C, Pan SJ, Wong E. The innate immunity protein IFITM3 modulates \u0026gamma;-secretase in Alzheimer\u0026rsquo;s disease. Nature. 2020 Oct 29;586(7831):735-40.\u003c/li\u003e\n\u003cli\u003eTamargo-G\u0026oacute;mez I, Mari\u0026ntilde;o G. AMPK: regulation of metabolic dynamics in the context of autophagy. International journal of molecular sciences. 2018 Nov 29;19(12):3812.\u003c/li\u003e\n\u003cli\u003eDeleyto-Seldas N, Efeyan A. The mTOR\u0026ndash;autophagy axis and the control of metabolism. Frontiers in cell and developmental biology. 2021 Jul 1;9:655731.\u003c/li\u003e\n\u003cli\u003eLin SC, Hardie DG. AMPK: sensing glucose as well as cellular energy status. Cell metabolism. 2018 Feb 6;27(2):299-313.\u003c/li\u003e\n\u003cli\u003eKang R, Zeh HJ, Lotze MT, Tang D. The beclin 1 network regulates autophagy and apoptosis. Cell Death \u0026amp; Differentiation. 2011 Feb 11;18(4):571-580.\u003c/li\u003e\n\u003cli\u003eGowans GJ, Hardie DG. AMPK: a cellular energy sensor primarily regulated by AMP. Biochemical Society Transactions. 2014 Jan 23;42(1):71-75.\u003c/li\u003e\n\u003cli\u003eHardie DG. AMP-activated protein kinase\u0026mdash;an energy sensor that regulates all aspects of cell function. Genes \u0026amp; development. 2011 Sep 15;25(18):1895-908.\u003c/li\u003e\n\u003cli\u003eCamacho-Castillo L, Phillips-Farf\u0026aacute;n BV, Rosas-Mendoza G, Baires-Lopez A, Toral-Rios D, Campos-Pena V. Increased oxidative stress contributes to enhance brain amyloidogenesis and blunts energy metabolism in sucrose-fed rat: Effect of AMPK activation. Scientific Reports. 2021 Oct 1;11(1):19547.\u003c/li\u003e\n\u003cli\u003eMasenga SK, Kabwe LS, Chakulya M, Kirabo A. Mechanisms of oxidative stress in metabolic syndrome. International journal of molecular sciences. 2023 Apr 26;24(9):7898.\u003c/li\u003e\n\u003cli\u003eIsrael MA, Yuan SH, Bardy C, Reyna SM, Mu Y, Herrera C, Hefferan MP, Van Gorp S, Nazor KL, Boscolo SF, Carson CT, Laurent LC, Marsala M, Gage FH, Remes AM, Koo EH, Goldstein LS. Probing sporadic and familial Alzheimer\u0026rsquo;s disease using induced pluripotent stem cells. Nature, 2012; 482(7384), 216-220.\u003c/li\u003e\n\u003cli\u003ePiaceri I, Nacmias B, Sorbi S. Genetics of familial and sporadic Alzheimer\u0026rsquo;s disease. Frontiers in Bioscience-Elite, 2023 Jan 1;5(1):167-177.\u003c/li\u003e\n\u003cli\u003eQuan M, Cao S, Wang Q, Wang S, Jia J. Genetic phenotypes of Alzheimer\u0026rsquo;s disease: Mechanisms and potential therapy. Phenomics, 2023 Apr 3;3(4):333-349.\u003c/li\u003e\n\u003cli\u003eKwon D, Moon BK, Han M, Lee TW, Lee J, Kang KS. Genetically stable multi-gene edited iPSCs-derived NK cells for enhanced cancer immunotherapy. Molecular Therapy Oncology, 2024; 32.4.\u003c/li\u003e\n\u003cli\u003eRim TA, Park N, Nam Y, Ham DS, Kim JW, Ha HY, Jung JW, Jung SM, Baek IC, Kim SY, Kim TG, Song J, Lee J, Park SH, Chung NG, Yoon KH, Ju JH. Recent progress of national banking project on homozygous HLA‐typed induced pluripotent stem cells in South Korea. Journal of tissue engineering and regenerative medicine. 2018 Dec 3; e1531-e1536.\u003c/li\u003e\n\u003cli\u003eTang D, Chen M, Huang X, Zhang G, Zeng L, Zhang G. SRplot: A free online platform for data visualization and graphing. PLoS One. 2023 Nov 9;18(11):e0294236.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer's Disease, Induced Pluripotent Stem Cells, Brain Organoid, Nano Graphene Oxide, Autophagy","lastPublishedDoi":"10.21203/rs.3.rs-8383146/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8383146/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground\u003c/p\u003e\n\u003cp\u003eAlzheimer’s disease (AD) is one of the best-known neurodegenerative diseases, and substantial progress has been made in the field of neuroscience and AD. However, there has been no major improvement in AD treatment.\u003c/p\u003e\n\u003cp\u003eMethods\u003c/p\u003e\n\u003cp\u003eCerebral organoids were generated using induced pluripotent stem cells (iPSCs) from both healthy individual and AD patients. Organoids were cultured for 12 weeks and then nano-graphene oxide (NGO) was treated for another 2 weeks. Organoids were then sampled according to different sampling methods for various analysis.\u003c/p\u003e\n\u003cp\u003eResults\u003c/p\u003e\n\u003cp\u003eAD patient-derived iPSCs were employed to generate cerebral brain organoids and were cultured until they expressed AD phenotypes, accumulation of amyloid-beta (Aβ) plaquesand hyperphosphorylated tau (pTau)-derived neurofibrillary tangles (NFTs). We investigated whether NGO treatment could decrease the expression of the major hallmarks of AD.\u003c/p\u003e\n\u003cp\u003eOur study illustrates that accumulated Aβ plaques were diminished in the NGO-treated AD organoids. NGO activates the autophagy pathway, targeting AMPK activation. The changes in the levels of AMP and ADP in NGO treated group suggest that NGO affects cellular energy metabolism, thus activating AMPK autophagic pathway. Also, the astrocytes and microglial cell presence in our model allowed examining further into inflammatory effect of NGO. The decrease in proinflammatory cytokine levels in NGO treated group led to decrease in IFITM3 expression and going further into diminishing γ-secretase activity.\u003c/p\u003e\n\u003cp\u003eConclusions\u003c/p\u003e\n\u003cp\u003eOverall, the development of AD brain organoids successfully mimickedAD phenotype expression;thus, they could be used as a screening platform for novel AD treatment assessments.\u003c/p\u003e","manuscriptTitle":"Autophagy and neuroinflammation modulation by nano-graphene oxide in PSEN1 Alzheimer’s brain organoids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 15:06:56","doi":"10.21203/rs.3.rs-8383146/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-11T00:49:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-03T06:29:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-23T14:35:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"249479881656062498998667566129423266202","date":"2026-02-23T13:44:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-20T16:10:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99403104990021854005831900980656299973","date":"2026-02-19T21:23:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"297370285671632873464268420340319750468","date":"2026-02-19T20:43:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"177486728317051931741909027054120053972","date":"2026-02-19T15:55:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154903557557309543541564760200691735301","date":"2026-02-19T07:59:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286993602231698663944792452315047193473","date":"2026-02-19T01:08:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"160644544394894833201651406734008342545","date":"2026-02-19T00:46:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-19T00:39:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-28T12:20:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-15T00:35:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2026-01-13T07:31:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b02b91af-bb20-4605-b2a4-2aae0bd93cda","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-17T20:08:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 15:06:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8383146","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8383146","identity":"rs-8383146","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}