extfieldsofstudy: Biology Medicine

text:
Correlation between miRNA target site polymorphisms in the 3′ UTR of AVPR1A and the risk of hypertension in the Chinese Han population

We aimed to study the relationship between rs11174811 and rs3803107 single nucleotide polymorphisms (SNPs) in miRNA target sites of the 3′ UTR in the arginine vasopressin receptor 1a gene (AVPR1A) and the risk of hypertension in the Chinese Han population. The genotypes at rs11174811 and rs3803107 were analyzed by direct sequencing in 425 Chinese Han patients with hypertension and 425 healthy subjects. AVPR1A expression was investigated by transfecting miR-526b, miR-375, and miR-186 mimics into human umbilical vein endothelial cells (HUVECs) containing AVPR1A rs11174811 CC, CA/AA and AVPR1A rs3803107 GG, GA/AA genotypes. The A alleles of rs11174811 (adjusted OR = 1.424, 95% CI: 1.231–1.599, P<0.001) and rs3803107 (adjusted OR = 1.222, 95% CI: 1.092–1.355; P=0.001) were high risk factors for hypertension. Plasma levels of miR-526b, miR-375, and miR-186 were higher in the study group than in the control group (P<0.001). The expression levels of AVPR1A mRNA in AVPR1A rs11174811 and rs3803107 mutant HUVECs were higher than those in wild-type cells (t = 8.811, 4.068 and P=0.001, 0.015, respectively). The single nucleotide polymorphisms rs11174811 and rs3803107 in the AVPR1A gene are associated with an increased risk of hypertension in the Chinese Han population. This may be related to the effect of these variants on the regulation of AVPR1A expression by miRNAs.

Introduction
Essential hypertension is a common cardiovascular disease in middle-aged and elderly populations. Its clinical characteristics are elevated blood pressure and multiple cardiovascular crises, which seriously affect the physical and mental health of patients and can lead to numerous complications and death [1].
Arginine vasopressin (AVP) is a vasoconstricting neuroendocrine hormone secreted by the supraventricular nucleus and paraventricular ventricles. AVP interacts with its receptor, AVPR1A, to cause the contraction of peripheral blood vessels [2,3]. Studies have shown that AVP levels are significantly elevated in the plasma of patients with heart failure and AVP can induce myocardial fibroblast proliferation and promote myocardial fibrosis through its V1a receptor [4,5].
The AVPR1A gene, encoding a receptor for AVP, is located at 12q14.2. It belongs to a subfamily of G protein-coupled receptors that also includes, AVPR1B, V2R, and oxytocin receptor [2]. AVPR1A activity is mediated by G proteins, which stimulate the phosphatidylinositol-calcium second messenger system [6]. The receptor affects cell contraction and proliferation, platelet aggregation, the release of coagulation factors, and glycogenolysis [7]. AVPR1A gene polymorphisms have become a hot research topic in recent years. The rs11174811 single nucleotide polymorphism (SNP) in the 3 UTR of AVPR1A has been associated with increased arterial blood pressure [8]. Variation at the rs3803107 locus of the AVPR1A gene is more common in the Chinese Han population than other populations, with a minimum allele frequency (MAF) as high as 0.1650 (Table 1). Both rs11174811 and rs3803107 are located in the 3 UTR region of AVPR1A, in the target sites for miRNAs. TargetScan 3.1 analysis predicted that the miR-526b target region was located at the rs11174811 locus, while the miR-375 and miR-186 target regions were located at the rs3803107 locus.
In the present study, we analyzed the relationship between the SNPs, rs11174811, and rs3803107 in miRNA target sites within the AVPR1A 3 UTR, and the risk of hypertension in the Chinese Han population. The role of miRNAs in regulating AVPR1A expression was also investigated.

Materials and methods Subjects
A total of 425 patients with ER, who were admitted to Zhuji People's Hospital of Zhejiang Province and Yidu Central Hospital of Weifang from June 2015 to December 2017, were randomly selected. This group of patients included 276 males and 149 females, ranging in age from 42-85 years, with an average age of 59.28 + − 7.84 years. The diagnostic criteria were: sitting systolic blood pressure (SBP) ≥140 mmHg and/or diastolic blood pressure (DBP) ≥90 mmHg [9]. Patients were excluded from the study if they had secondary hypertension, diabetes, coronary heart disease, valvular heart disease, and other organic heart disease, cancer, or immune system diseases. A control group of 425 healthy subjects was recruited, with matching age and gender to those of the patients in the study group. Control subjects ranged in age from 41-85 years, with a mean age of 60.82 + − 13.45 years. Subjects were included in the control group if they met the following criteria: SBP <140 mmHg; DBP <90 mmHg; and the absence of diabetes, coronary heart disease, organic heart disease, cancer, and immune system diseases. The present study was approved by the Medical Ethics Committee of Zhuji People's Hospital of Zhejiang Province and Yidu Central Hospital of Weifang and all subjects gave informed consent.

Genotyping
Venous blood (5 ml) was collected from all subjects, plasma cells were separated, and genomic DNA was extracted using a QIAamp DNA Blood Mini Kit (Qiagen, Venlo, Netherlands). Genotypes at the rs11174811 and rs3803107 loci in the AVPR1A gene's 3 UTR were detected using Sanger sequencing technology. Sequencing primers for rs11174811 (F: 5 -AGG CCA CTG CCA GTT GTA AA-3 ; R: 5 -GAG TAC AAG TGC CTG GGG TG-3 ) and rs3803107 (F: 5 -AGT GCC GCA TTT TAT GTG ACT-3 ; R: 5 -ATG CAG GTC TGA TTC CCA GA-3 ) were designed based on the published chromosomal location of these SNPs (Table 1). Representative sequencing results for each genotype are shown in Figure 1.

miRNA expression analysis
Plasma miRNAs were extracted using a PureLink RNA Mini Kit™ (catalog number 12183018A, Invitrogen, Carlsbad, CA, U.S.A.). miRNA was reverse transcribed into cDNA using a reverse transcription kit (TOYOBO, Osaka, Japan) and reverse transcription primers were purchased from Guangzhou Ribo Bio Co. Ltd (Guangzhou, China). U6 RNA was used as a miRNA control. Quantitative real-time PCR was performed using a SYBR-Green PCR kit (catalog number 208054, TOYOBO), with three replicate wells per sample. miRNA expression level was analyzed using the 2 − C T method.

Quantitative real-time PCR
Total RNA was extracted from collected cells using an RNeasy Mini Kit (Qiagen) and reverse transcribed into cDNA using a reverse transcription kit (TOYOBO). AVPR1A was amplified using the following primers: forward, 5 -CTTTTGTGATCGTGACGGCTTA-3 and reverse, 5 -TGATGGTAGGGTTTTCCGATTC-3 . GAPDH was used as an internal reference and amplified using the following primers: forward, 5 -ACCCAGAAGACTGTGGATGG-3 and reverse, 5 -TTCTAGACGGCAGGTCAGGT-3 . qRT-PCR was performed using a SYBR-Green PCR kit (TOY-OBO), with three replicate wells per sample. miRNA expression level was analyzed using the 2 − C T method. The reaction mixture comprised 10 μl of SYBR Green Real Master Mix (Promega), 1.6 μl of primer mix (200 nM), and 1.6 μl of cDNA template. The thermal cycling conditions were as follows: 3 min at 95 • C for denaturation, then 38 cycles of 95 • C for 10 s, 60 • C for 30 s, and 72 • C for 30 s. After the PCR was completed, a melting curve analysis was performed to ensure the purity of the PCR product.

Statistical analysis
Continuous variables are expressed as mean + − S.D. (x + − s) and comparisons between groups were performed using an independent sample Student's t test. Categorical variables are expressed as a percentage (n[%]) and comparisons between groups were performed using a χ 2 test. A χ 2 test was also used to determine whether genotype frequencies were consistent with those at Hardy-Weinberg equilibrium (HWE). The association of SNPs with hypertension risk was determined based on the distribution of allele frequencies and genetic models (dominant and recessive models).  Odds ratios (OR) and 95% CI were used in unconditional logistic regression analysis, with corrections for age, sex, body mass index (BMI), smoking, drinking, and family history of hypertension. Statistical analysis was performed using SPSS21.0 software (IBM, Chicago, IL, U.S.A.). All tests were two-tailed and differences were considered statistically significant at P<0.05.

Demographic characteristics
There were no significant differences in age or gender between the study group and the control group (P>0.05). BMI, smoking, drinking, and a family history of hypertension were all more frequent in the study group than in the control group. DBP and SBP were also significantly higher in the study group than in the control group (P<0.05, Table 2).

AVPR1A 3 UTR SNPs and risk of hypertension
The genotype distributions of the rs11174811 and rs3803107 loci were consistent with their frequencies at HWE (P=0.252, 0.274, respectively). Using the wild type (CC) of the rs11174811 locus as a reference, the risk of hypertension was increased in homozygous, dominant, and recessive models (adjusted OR = 1.927, 95% CI:  The criterion for smoking was: smoking more than five cigarettes a week. The criterion for drinking was: consumption of alcohol more than three times a year, in excess of 50 mL each time.

Stratified analysis of AVPR1A 3 UTR SNPs and risk factors for hypertension
The stratified analysis of age, sex, BMI, smoking status, drinking status, and family history of hypertension showed that the risk of hypertension was increased in subjects with the AVPR1A rs11174811 mutation (CA/AA) if they also had an age >60 years (adjusted OR =  Table 4). The risk of hypertension was significantly increased in subjects with the AVPR1A rs3803107 mutation (GA/AA) only if they had a family history of hypertension (adjusted OR = 1.262; 95% CI: 1.006-1.514; P=0.044; Table 5). The criterion for smoking was: smoking more than five cigarettes a week. The criterion for drinking was: consumption of alcohol more than three times a year, in excess of 50 ml each time.

Plasma miRNA expression levels
Plasma levels of miR-526b, miR-375, and miR-186 were significantly higher in the study group than in the control group (P<0.001, Figure 2). There was no difference in plasma miRNA-526b levels between subjects with wild type (CC) or mutant (CA/AA) genotypes at the rs11174811 locus (P=0.228, Figure 3A). Likewise, there was no difference in plasma miR-375 and miR-186 levels between subjects with wild-type (GG) or mutant (GA/AA) genotypes at the rs3803107 locus (P=0.355 and 0.377, respectively; Figure 3B,C). The criterion for smoking was: smoking more than five cigarettes a week. The criterion for drinking was: consumption of alcohol more than three times a year, in excess of 50 ml each time.

The correlation between AVPR1A 3 UTR SNPs and plasma miRNA levels
The effect of the rs11174811 and rs3803107 genotypes on AVPR1A mRNA expression was investigated. AVPR1A mRNA expression level was higher in cells with mutant genotypes at rs11174811 (t = 8.811, P=0.001) and rs3803107 (t = 4.068; P=0.015) loci than in wild-type cells ( Figure 4A,B). AVPR1A mRNA expression increased in HU-VECs after transfection with miR-526b, miR-375, and miR-186 (P<0.05, Figure 4C-E). Western blotting showed that AVPR1A protein expression level was also higher in mutant cells than in wild-type cells ( Figure 5).

Discussion
In the present study, we analyzed the correlation between rs11174811 and rs3803107 SNPs in the 3 UTR of the AVPR1A gene and the risk of hypertension in the Chinese Han population. Our results showed that carriers of the A allele at the rs11174811 locus had a 1.424 times higher risk of developing hypertension than carriers of the C allele (95% CI: 1.231-1.599, P<0.001). The rs3803107 locus A allele was associated with a 1.222 times higher risk of hypertension than the G allele (95% CI: 1.092-1.355, P=0.001). Furthermore, rs11174811 and rs3803107 mutant AVPR1A were expressed at a higher level than wild-type AVPR1A in HUVECs. Therefore, we believe that these SNPs in the 3 UTR of the AVPR1A gene affect miRNA-mediated regulation of AVPR1A gene expression. As the population ages, the incidence of hypertension in China is gradually increasing. Hypertension is an independent risk factor for cardiovascular and cerebrovascular diseases, which are important causes of mortality and disability in the elderly. Therefore, the prevention and treatment of hypertension in the elderly are especially important [10,11]. The development of hypertension is caused by multiple factors, including the interaction of genetic and environmental factors [12,13]. During the development of hypertension, the renin-angiotensin-aldosterone system, the sympathetic nervous system, and ion transporters in the kidneys and other tissues play important roles [14][15][16] In recent years, a correlation between non-coding RNAs (ncRNAs) and human diseases has been reported. There have been many studies on the relationship between ncRNAs and cardiovascular diseases or hypertension and numerous miRNAs have been shown to have an important role in the process of hypertension [17][18][19]. Studies have found that the rs11174811 SNP in an miRNA binding site within the AVPR1A gene, and is correlated with elevated blood pressure and risk of myocardial infarction in Caucasians [20]. In addition, the researchers found that this SNP was not correlated with elevated blood pressure in a southern Indian population [8]. This suggests that the correlation between rs11174811 and hypertension risk may be related to ethnicity. The subjects in the present study were Han Chinese from southern China. A total of 425 hypertensive patients were included, which is a relatively small number. We found that the MAF (A) at the rs11174811 locus was 4.71%, while data from the 1000 Genomes Project database showed that the frequency of this allele was 4.85% in the Han population from Beijing, China and 2.86% in the Han population from southern China. This indicates that the subjects selected in the present study are representative of the population and the results are highly reliable. The rs3803107 locus is also located in the 3 UTR region of AVPR1A, but few studies have focussed on this site. Tansey et al. [21] studied four SNPs in AVPR1A (rs3803107, rs1042615, rs3741865, and rs11174815) and three microsatellites (RS3, RS1, and AVR) to assess their association with autism in an Irish population. They found that, out of these four SNPs, only rs11174815 was correlated with autism. The results of the present study showed that rs3803107 was correlated with hypertension risk in a Chinese Han population.
rs11174815 and rs3803107 are located in the 3 UTR of AVPR1A. TargetScan predicted that the miR-526b target site is located at rs11174815, and the miR-375 and miR-186 target sites are located at rs3803107. Plasma levels of miR-526b, miR-375, and miR-186 were higher in hypertensive patients than in healthy subjects (P<0.001). The data in the present study are not sufficient to explain the increase in the plasma levels of miRs in hypertensive patients, which may be due to a combination of factors, such as long-chain ncRNA expression regulation, which is an interesting research direction. Therefore, we speculate that the regulation of AVPR1A expression by miR-526b, miR-375, and miR-186 may be related to the occurrence of hypertension. In order to confirm our hypothesis, we used CRISPR/Cas9 technology to construct HUVECs with CC or CA/AA genotypes at rs11174811 and HUVECs with GG or GA/AA genotypes at rs3803107. After transfection of miR-526b, miR-375, and miR-186 mimics, AVPR1A mRNA expression level increased, indicating that these miRNAs can regulate the expression of AVPR1A, which was consistent with the results in hypertensive patients. Analysis of the expression levels from different genotypes in transfected cells showed that the expression level of AVPR1A mRNA in the mutant cells was significantly higher than that in the wild-type cells. The results of western blotting showed that AVPR1A protein expression was also significantly higher in the mutant cells than in wild-type cells. From these results, it can be concluded that the rs11174811 and rs3803107 SNPs affect the regulation of AVPR1A expression by miRNAs, which may be related to the effect of sequence variation on the binding efficiency of miRNAs to their target sites. Human genes can be regulated by a variety of miRNAs [22,23] and genetic variation at miRNA target sites in the 3 UTR of genes affects miRNA target recognition [24]. In addition, in the present study, the plasma levels miR-526b, miR-375, and miR-186 were not correlated with the genotypes at rs11174811 and rs3803107 (P>0.05). This suggests that miRNA target recognition efficiency is key to determining the expression level of AVPR1A in the presence of the same level of miRNA. Although we do not have direct evidence to support the inhibition of AVPR1A expression by miRNA via binding to AVPR1A mRNA, it may also have involved other mechanisms and more direct evidence is needed. The expression of AVPR1A was not as expected when transfected with 100 ng of ssEGFP miRNA in vitro, and there may be other unknown reasons, but the difference in AVPR1A expression levels between the different genotypes was consistent with our expectations.
There are some shortcomings in the present study. First, the sample size was insufficient, the number of homozygotes was small, and the analysis error was large. Therefore, we combined heterozygotes and homozygotes in the analysis. Second, there may be some differences between the in vitro cell model used here and the regulation of miRNA expression in vivo. In addition, the occurrence of hypertension may involve a complex effect of multiple genes and non-genetic factors. The role of a single gene may be relatively minor and further research is needed. study design, the analysis and interpretation of data, and preparation of the manuscript. All authors read and approved the final manuscript.


extfieldsofstudy: Medicine Chemistry

text:
On-Membrane Dynamic Interplay between Anti-GM1 IgG Antibodies and Complement Component C1q

Guillain–Barré syndrome, an autoimmune neuropathy characterized by acute limb weakness, is often preceded by Campylobacter jejuni infection. Molecular mimicry exists between the bacterial lipo-oligosaccharide and human ganglioside. Such C. jejuni infection induces production of immunoglobulin G1 (IgG1) autoantibodies against GM1 and causes complement-mediated motor nerve injury. For elucidating the molecular mechanisms linking autoantigen recognition and complement activation, we characterized the dynamic interactions of anti-GM1 IgG autoantibodies on ganglioside-incorporated membranes. Using high-speed atomic force microscopy, we found that the IgG molecules assemble into a hexameric ring structure on the membranes depending on their specific interactions with GM1. Complement component C1q was specifically recruited onto these IgG rings. The ring formation was inhibited by an IgG-binding domain of staphylococcal protein A bound at the cleft between the CH2 and CH3 domains. These data indicate that the IgG assembly is mediated through Fc–Fc interactions, which are promoted under on-membrane conditions due to restricted translational diffusion of IgG molecules. Reduction and alkylation of the hinge disulfide impaired IgG ring formation, presumably because of an increase in conformational entropic penalty. Our findings provide mechanistic insights into the molecular processes involved in Guillain–Barré syndrome and, more generally, into antigen-dependent interplay between antibodies and complement components on membranes.

Introduction
Immunoglobulin G (IgG) is a crucial mediator of the defensive mechanisms for eliminating infectious microorganisms [1]. Antigenic determinants displayed on the surfaces of invasive cells are recognized by host IgG antibodies, which trigger effector functions, such as complement-mediated cytotoxicity and opsonic phagocytosis. To evade the host immune system, mechanisms have evolved Int. J. Mol. Sci. 2020, 21, 147 2 of 12 in infectious bacteria to coat their surfaces with molecules derived from infected hosts or molecules mimicking those presented on host cells. Some Gram-positive bacteria possess cell wall proteins capable of binding to IgG molecules independent of their antigen-binding sites. Examples of such proteins are protein A from Staphylococcus aureus and protein G from streptococcus groups C and G, which can disturb host immune mechanisms [2,3]. In contrast, some Gram-negative bacteria can express outer membrane glycolipids that share common glycan structures with mammalian glycosphingolipids, enabling them to escape the immune surveillance [4,5]. However, such bacterial glycolipids occasionally elicit the production of antibodies that are cross-reactive with host molecules [6]. Therefore, the molecular mimicry between components of infectious bacteria and the host has been postulated as the mechanism underlying the onset and development of autoimmune diseases.
Guillain-Barré syndrome is a post-infectious autoimmune neuropathy characterized by acute limb weakness. One-third of patients with Guillain-Barré syndrome are preceded by Campylobacter jejuni enteritis [7]. Molecular mimicry exists between two-thirds of C. jejuni strains and human ganglioside GM1, which is a glycosphingolipid highly expressed at the nodal membranes of human motor nerves. In one out of one thousand individuals, such C. jejuni infection could induce the production of anti-GM1 IgG1 autoantibodies and consequent complement-mediated motor nerve injury, causing limb weakness [8]. However, the molecular mechanisms linking autoantigen recognition and complement activation remain largely unknown.
To gain mechanistic insights into the molecular process behind these interactions, we attempted to observe the dynamic interactions of an anti-GM1 monoclonal autoantibody, GB2. This autoantibody was generated by immunization of mice with GM1-like lipo-oligosaccharide purified from a C. jejuni strain isolated from a patient with Guillain-Barré syndrome [6]. In a previous study, we demonstrated that high-speed atomic force microscopy (HS-AFM) is a powerful tool for real-time observation of interactions of IgG molecules with the Fcγ receptor [9]. Here we applied this technique for visualizing the interplay between GB2 and complement component C1q on membranes containing GM1, which is the first step of the classical complement pathway in the immune system.

Epitope Mapping of GB2
Previous studies revealed that GB2, directed against C. jejuni, was cross-reactive with GM1 but not with the other ganglioside components in the bovine brain, e.g., GD1a, GD1b, and GT1b [6], nor synthesized gangliosidic oligosaccharides derived from GM3, GD3, GM2, GD1a, and GT1a [10]. We conducted a saturation transfer difference (STD) NMR experiment using a synthetic GM1 pentasaccharide for characterizing antigen recognition by GB2. In this experiment, the saturated magnetization of antibody protons can be transferred to the oligosaccharide in their bound state, thereby enabling the identification of the carbohydrate epitope recognized by GB2 based on STD data. The STD data indicated that the common carbohydrate moiety shared with C. jejuni lipo-oligosaccharide was extensively involved in the interaction with the antibody GB2, which explained its highly specific interaction with GM1 (Figure 1a,b). Our STD data obtained by using a GM2 tetrasaccharide also confirmed that the interaction was significantly compromised by the removal of the outer galactose residue. In the subsequent experiments, we characterized the interaction of GB2 with GM1 in membrane environments (Figure 1c,d). (c) Overlay of the 2D 1 H-13 C HSQC spectrum (black) and the 1 H-13 C STD-HSQC spectrum (red) of the 2-trimethylsilylethyl derivatives of the GM2 tetrasaccharide (10 equiv.) with 50 μM GB2. (d) Mapping of the binding epitope of the GM2 tetrasaccharide. In (b,d), the maximum peak intensity of each oligosaccharide was taken as 100%, and the relative intensities (over 80% red, over 65% pink, over 50% light pink) are indicated. R represents a 2-trimethylsilylethyl group.

IgG Assembly on Antigen-Incorporated Membranes
For HS-AFM observation of the molecular behavior of GB2 on a membrane, a mica surface was covered with a lipid bilayer composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and GM1 in varying ratios. The GM1/DOPC lipid had a uniform surface but not phase-separated domains (Supplementary Figure S1a). In addition, we confirmed that GM1 molecules were uniformly distributed in the membrane as probed with cholera toxin B subunit, a GM1-specific binder (Supplementary Figure S1b). In the absence of GM1, we could not see obvious spots of the IgG molecules bound to the membrane. This is because the affinity of IgG for DOPC was so weak that IgG diffused much faster than the imaging speed of HS-AFM. By contrast, binding of the IgG molecules assembled into well-ordered hexameric ring structures, besides larger aggregates and assembly intermediates, was clearly observed on the membranes with higher (>25%) GM1 ratios ( Figure 2, Supplementary Movie S1-3). When GM2 was used instead of GM1, the IgG molecules flitted on the membrane but never formed the ring structure ( Figure 2f, Supplementary Movie S4). These results indicate that antigen binding promotes the hexameric ring formation of the GB2 antibodies on the membrane. (c) Overlay of the 2D 1 H-13 C HSQC spectrum (black) and the 1 H-13 C STD-HSQC spectrum (red) of the 2-trimethylsilylethyl derivatives of the GM2 tetrasaccharide (10 equiv.) with 50 µM GB2. (d) Mapping of the binding epitope of the GM2 tetrasaccharide. In (b,d), the maximum peak intensity of each oligosaccharide was taken as 100%, and the relative intensities (over 80% red, over 65% pink, over 50% light pink) are indicated. R represents a 2-trimethylsilylethyl group.

IgG Assembly on Antigen-Incorporated Membranes
For HS-AFM observation of the molecular behavior of GB2 on a membrane, a mica surface was covered with a lipid bilayer composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and GM1 in varying ratios. The GM1/DOPC lipid had a uniform surface but not phase-separated domains (Supplementary Figure S1a). In addition, we confirmed that GM1 molecules were uniformly distributed in the membrane as probed with cholera toxin B subunit, a GM1-specific binder (Supplementary Figure S1b). In the absence of GM1, we could not see obvious spots of the IgG molecules bound to the membrane. This is because the affinity of IgG for DOPC was so weak that IgG diffused much faster than the imaging speed of HS-AFM. By contrast, binding of the IgG molecules assembled into well-ordered hexameric ring structures, besides larger aggregates and assembly intermediates, was clearly observed on the membranes with higher (>25%) GM1 ratios ( Figure 2, Supplementary Movies S1-S3). When GM2 was used instead of GM1, the IgG molecules flitted on the membrane but never formed the ring structure ( Figure 2f, Supplementary Movie S4).
These results indicate that antigen binding promotes the hexameric ring formation of the GB2 antibodies on the membrane.

C1q Interaction with the IgG Hexameric Ring Formed on Membranes
Next, we attempted to address the pathological relevance of the antigen-dependent IgG hexamer formation. We examined the potential interaction of the IgG hexameric ring with C1q using HS-AFM, because accumulating data have indicated that C1q preferentially binds to IgG oligomers [11][12][13][14]. HS-AFM could clearly visualize dynamic structures of free C1q molecules on mica surface treated

C1q Interaction with the IgG Hexameric Ring Formed on Membranes
Next, we attempted to address the pathological relevance of the antigen-dependent IgG hexamer formation. We examined the potential interaction of the IgG hexameric ring with C1q using HS-AFM, because accumulating data have indicated that C1q preferentially binds to IgG oligomers [11][12][13][14]. HS-AFM could clearly visualize dynamic structures of free C1q molecules on mica surface treated with 3-aminopropyltriethoxysilane, exhibiting six highly mobile globular heads tethered to the central stem, whereas C1q molecules were not clearly observable on the membranes because of rapid diffusion due to weak interaction with the membrane surface (Figure 3a

Effect of Protein A Binding and Disulfide Cleavage on IgG Hexameric Ring Formation
The bacterial IgG-binding proteins, protein A and protein G, have been reported to inhibit C1q binding to IgG antibodies [2,15]. Our HS-AFM data showed that GB2 scarcely formed the hexameric ring structure in the presence of the B domain of protein A, which is known to bind the Fc region of IgG antibodies [2,[16][17][18] (Figure 4). We solved the crystal structures of the Fc fragment of GB2 and

Effect of Protein A Binding and Disulfide Cleavage on IgG Hexameric Ring Formation
The bacterial IgG-binding proteins, protein A and protein G, have been reported to inhibit C1q binding to IgG antibodies [2,15]. Our HS-AFM data showed that GB2 scarcely formed the hexameric ring structure in the presence of the B domain of protein A, which is known to bind the Fc region of IgG antibodies [2,[16][17][18] (Figure 4). We solved the crystal structures of the Fc fragment of GB2 and that of the same IgG isotype in the absence or presence of the B domain of protein A, respectively. In the crystal, the apo form of Fc formed a trimer but not a hexamer, confirming that the ring formation is an on-membrane process mediated by antigen-Fab interaction (Supplementary Figure S2). Our crystallographic data also confirmed that, as in the case of previously reported crystal structures [17,18], the Fc interacts with the B domain of protein A at the cleft between the C H 2 and C H 3 domains, which is also involved in protein G binding [19,20] (Figure 4e, Supplementary  Figure S3). These data indicate that the antigen-dependent hexameric ring formation of IgG is mediated by Fc-Fc interactions, which is disrupted by competitive binding with protein A.
Int. J. Mol. Sci. 2020, 20, x FOR PEER REVIEW 6 of 12 that of the same IgG isotype in the absence or presence of the B domain of protein A, respectively. In the crystal, the apo form of Fc formed a trimer but not a hexamer, confirming that the ring formation is an on-membrane process mediated by antigen-Fab interaction (Supplementary Figure S2). Our crystallographic data also confirmed that, as in the case of previously reported crystal structures [17,18], the Fc interacts with the B domain of protein A at the cleft between the CH2 and CH3 domains, which is also involved in protein G binding [19,20] (Figure 4e, Supplementary Figure S3). These data indicate that the antigen-dependent hexameric ring formation of IgG is mediated by Fc-Fc interactions, which is disrupted by competitive binding with protein A. Complement activation was reported to be impaired by the reduction and alkylation of disulfide bonds in the hinge region of IgG antibodies [21]. We examined the possible impact of the cleavage of IgG interchain disulfide bridges on the antigen-dependent assembly of the GB2 autoantibodies. The results indicate that the disulfide cleavage causes a significant reduction in the GB2 hexamer formation (Figure 4c,d).

Discussion
The anti-GM1 antibody GB2 was found to stain nodes and axons in the motor nerve roots in rabbit models of Guillain-Barré syndrome [6]. Anti-GM1 IgG antibodies cause complementmediated membrane disruption at nodes of Ranvier in peripheral motor nerve fibers in humans and Complement activation was reported to be impaired by the reduction and alkylation of disulfide bonds in the hinge region of IgG antibodies [21]. We examined the possible impact of the cleavage of IgG interchain disulfide bridges on the antigen-dependent assembly of the GB2 autoantibodies.
The results indicate that the disulfide cleavage causes a significant reduction in the GB2 hexamer formation (Figure 4c,d).

Discussion
The anti-GM1 antibody GB2 was found to stain nodes and axons in the motor nerve roots in rabbit models of Guillain-Barré syndrome [6]. Anti-GM1 IgG antibodies cause complement-mediated membrane disruption at nodes of Ranvier in peripheral motor nerve fibers in humans and rabbits [22,23]. Our study visualized antigen-dependent assembly of the GB2 antibodies on model membranes, giving rise to hexameric ring structures, which particularly interacted with C1q.
In recent studies, the binding of C1q with IgG oligomers was studied, most notably by employing artificially hexamerized IgG antibodies [11][12][13][14]. It has been reported that IgG molecules have the potential ability to form hexamers in crystal or on mica surface under certain conditions [14,24]. In human IgG, the hexamer formation could be enhanced by triple mutation at the C H 3 domain (i.e., E345R, E430G, and S440Y), which improves the complement activating activity [14]. Cryo-electron microscopy data have revealed that C1q interacts with the hexamerized IgG, and each globular head of C1q is bound to the hinge-proximal C H 2 domains of IgG antibodies, which is distal from the binding sites of the bacterial IgG-binding proteins [12]. Intriguingly, the mouse anti-GM1 IgG2b(κ) antibody GB2, in which positions 345, 430, and 440 are occupied by alanine, glutamic acid, and threonine, respectively, were highly preorganized into the hexameric ring on the antigenic membrane formation without artificial mutations. These amino acid residues are not directly involved in the binding to the B domain of protein A, which, however, sterically inhibits Fc-Fc interaction. Therefore, the inhibition of C1q binding by protein A and protein G is, at least, partially attributed to the prevention of Fc-mediated ring formation.
In the wild-type IgG2b, the hexameric ring formation occurred exclusively on the membranes containing specific antigenic molecules and did not occur in solution. This ordering process is entropically unfavorable; therefore, it is promoted selectively under on-membrane conditions, where diffusion of IgG molecules is considerably restricted. Upon loss of the constraint of disulfide bonds at the hinge, IgG antibodies gain greater degrees of motional freedom of the C H 2 domains, with an increase in the population of extremely asymmetric quaternary conformations [25]. Therefore, the reduction and alkylation of the hinge disulfides of IgG causes an increase in the conformational entropic penalty for hexamerization, resulting in the impaired formation of IgG rings reactive with C1q.
In summary, our findings provide mechanistic insights into the molecular processes involved in Guillain-Barré syndrome and, more generally, into the antigen-dependent interplay between antibodies and complement components on membranes. It is possible that Fc-mediated hexameric ring formation depends not only on antigen type but also on IgG glycoforms because C1q binding to IgG is reported to depend on galactosylation and sialylation of Fc [26][27][28]. Our subsequent studies will address these issues, which will provide clues to control IgG assembly and the consequent complement activation by employing antibody engineering approaches.

Antibody
The mouse anti-GM1 IgG2b(κ) antibody GB2 was produced in mouse hybridoma cells [6], which were cultivated in the NYSF 404 serum-free medium (Nissui, Tokyo, Japan). After cell growth, the medium supernatant was applied onto an nProtein A Sepharose Fast Flow column (GE Healthcare, Chicago, IL, USA), followed by gel filtration using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare, Chicago, IL, USA) with phosphate-buffered saline consisting of 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 , and KH 2 PO 4 (pH 7.4) to purify GB2. Cleavage of the interchain disulfide bridges of GB2 was performed according to the literature [31]. For brevity, GB2 was reduced by 10 mM DTT at room temperature for 1 h in 1.5 M Tris-HCl, pH 8.5, containing 2 mM EDTA. For alkylation, 22 mM iodoacetic acid was added to the above reaction mixture, which was incubated in the dark for 20 min at room temperature. The GB2 antibody and its reduced and alkylated analog, thus prepared, were dialyzed against phosphate-buffered saline and were subjected to HS-AFM measurements. For X-ray crystallographic analysis, the Fc fragments were prepared from GB2 and mouse anti-progesterone IgG2b(κ) antibody 7D7 [32] by papain digestion, performed at 37 • C for 12 h in 75 mM sodium phosphate buffer (pH 7.0) containing 75 mM NaCl and 2 mM EDTA. The protein concentration was 10 mg/mL, and the ratio of papain:IgG was 1:50 (w:w). The digestion products were loaded onto an Affi-gel protein A column (Biorad, Berkely, CA, USA) to obtain purified Fc fragments.

Protein A
The cDNA of the B domain of protein A was purchased from Fasmac Co., Ltd. (Kanagawa, Japan) and subcloned into a pET28b expression vector (Merck KGaA, Darmstadt, Germany). A recombinant B domain with an N-terminal hexahistidine tag was expressed in Escherichia coli strain BL21(DE3)-CodonPlus (Stratagene, San Diego, CA, USA). The hexahistidine-tagged B domain was then purified using a Chelating Sepharose Fast Flow column (GE Healthcare, Chicago, IL, USA). Subsequently, the hexahistidine tag was cleaved using thrombin, followed by gel filtration chromatography using a HiLoad 16/60 Superdex75 (GE Healthcare, Chicago, IL, USA) column with 50 mM Tris-HCl, pH 8.0 containing 150 mM NaCl.

C1q
C1q was purified from 40 mL of pooled human serum (Cosmo Bio CO., LTD, Tokyo, Japan) via two-step precipitation at low ionic strength [33]. To 40 mL human serum, 10 mL of 0.1 M Na 2 EDTA solution (pH 7.5) was added to dissociate the C1 complex into its constitutive subunits. To precipitate C1q, 200 mL of 0.01 M Na 2 EDTA (pH 7.5) was added to the solution, which was then incubated on ice for 1 h. The precipitate was collected by centrifugation (12,000× g for 30 min, if not specified), washed with 0.04 M Na 2 EDTA (pH 7.5), and collected via centrifugation. The washing step was done twice. The precipitate was dissolved in 10 mL of 0.01 M Na 2 EDTA (pH 5.0) containing 0.75 M NaCl for further purification. The supernatant was collected via centrifugation at 30,000× g for 30 min and then dialyzed into 1 L of 0.1 M Na 2 EDTA (pH 5.0) twice. The precipitate was collected via centrifugation, washed in 20 mL of 0.1 M Na 2 EDTA (pH 5.0), and again collected via centrifugation. The washing step was done twice. The collected precipitate was dissolved in 3 mL of 0.01 M Na 2 EDTA (pH 7.5) containing 0.3 M NaCl, and the supernatant was again collected via centrifugation at 30,000× g for 30 min. The supernatant contained 0.2 mg/mL C1q.

X-Ray Crystallography
The apo Fc crystals derived from GB2 were obtained in a buffer containing 16% PEG3350, 0.2 M lithium chloride, and 0.1 M Bis-Tris (pH 6.5) after incubation at 20 • C for one week. The Fc fragment cleaved from 7D7 and the B domain of protein A were mixed at a molar ratio of 1:2.5 and then applied to a gel filtration column (Superose 75; GE Healthcare, Chicago, IL, USA) equilibrated with 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl. Fractions containing the Fc-B domain complex were concentrated to a total protein concentration of 10 mg/mL and used for crystallization. The crystals of Fc in complex with the B domain of protein A were obtained in 60% (v/v) Tacsimate TM (pH 7.0) incubated at 20 • C for 2 weeks. The crystals were cryoprotected with the crystallization buffer supplemented with 15% glycerol. The apo crystal belonged to space groups P2 1 2 1 2 1 with three Fc dimers per asymmetric unit and diffracted up to a resolution of 2.30 Å. On the other hand, the B-domain-bound Fc crystal belonged to space group P4 2 2 1 2, with an asymmetric unit in which one Fc dimer complexed with one B domain, and one Fc monomer formed a homodimer with the crystallographically neighboring Fc molecule and diffracted up to a resolution of 3.30 Å. Diffraction data were integrated and scaled using XDS and AIMLESS [35,36].
The apo and complex crystal structures were solved by the molecular replacement method using the program MOLREP [37] with an apo form of mouse IgG2b-Fc (Protein Data Bank code: 2RGS [38]) as a search model. Manual model fitting to the electron density maps was carried out using COOT [39]. REFMAC5 [40] and phenix.refine [41] were used to refine the crystal structure, and the stereochemical quality of the final model was validated using RAMPAGE [42] (The crystal parameters and refinement statistics are summarized in Supplementary Table S1). The molecular graphics were prepared using PyMOL (http://www.pymol.org/).

PDB Accession Codes
The coordinates and structural factors of the crystal structures of the apo and B-domain-bound forms of mouse IgG2b-Fc have been deposited in the Protein Data Bank under accession numbers 6KRU and 6KRV, respectively.

HS-AFM
Ganglioside (GM1 or GM2) and DOPC were mixed at varying ratios in methanol/chloroform. After solvent evaporation, the residual lipids were suspended at a concentration of 12 mM in 10 mM potassium phosphate buffer (pH 7.2) and then mixed by vortexing. The mixture was sonicated at 70 W for 1 min. The liposome solution of approximately 2 µL was deposited onto a freshly cleaved mica surface and stored at 50 • C in a sealed container to maintain high humidity and prevent surface drying. After a 30-min incubation, the sample surface was thoroughly rinsed with pure water; then, a droplet of antibody solution in phosphate-buffered saline was deposited on the lipid-covered mica surface at a final protein concentration of 10 µg/mL and incubated for 3 min unless otherwise stated. Residual antibodies in the solution were removed by washing. Two molar equivalent of B domain of protein A was added to GB2 before applying to the mica surface. The HS-AFM experiments were conducted three times for each condition. C1q was added at a final concentration of 5-10 µg/mL for observing its interaction with GB2. The mica surface was chemically modified with 0.05% 3-aminopropyltriethoxysilane for observing C1q thereon.
All HS-AFM measurements were carried out using a laboratory-built AFM operated in tapping mode [43,44]. Small cantilevers (BL-AC7DS: Olympus, Tokyo, Japan) with a spring constant of 0.2 Nm −1 , a quality factor of approximately 2, and a resonant frequency of~0.8 MHz (all properties were estimated in water) were used. To achieve a small tip-sample loading force, the free oscillation amplitude of cantilevers was set at 1-2 nm, and the set-point of amplitude for feedback control was approximately 90% of the free amplitude. The HS-AFM experiments were performed at room temperature.
The bound-state dwell time was measured using successive HS-AFM images to estimate the residence time of C1q on the hexameric IgG ring by monitoring the appearance or disappearance of bright spots in the HS-AFM images. All analyses were carried out using laboratory-developed software based on IgorPro 8 (WaveMetrics, Inc., Lake Oswego, OR, USA).


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Modulation of RECK levels in Xenopus A6 cells: effects on MT1-MMP, MMP-2 and pERK levels

Background MT1-MMP is a cell-surface enzyme whose regulation of pro-MMP-2 and ERK activation position it as a key facilitator of ECM remodelling and cell migration. These processes are modulated by endogenous MMP inhibitors, such as RECK, a GPI-anchored protein which has been shown to inhibit both MT1-MMP and MMP-2 activity. Our previous studies have revealed a link between MT1-MMP levels, and pro-MMP-2 and ERK activation in mammalian cells, as well as MT1-MMP and RECK co-localization in Xenopus embryos. We here investigated how modulation of RECK would impact MT1-MMP and MMP-2 levels, as well as ERK signalling in Xenopus A6 cells. Results We used a Morpholino approach to knockdown RECK, plasmid transfection to overexpress RECK, and PI-PLC treatment to shed RECK from the cell surface of Xenopus A6 cells. RECK reduction did not alter pERK or MT1-MMP levels, nor MMP-2 activity as measured by zymography; thus RECK-knockdown cells maintained the ability to remodel the ECM. RECK overexpression and PI-PLC treatment both increased ECM remodelling potential through increased MT1-MMP protein and relative MMP-2 activation levels. Conclusions RECK changes that reduce the ability of the cell to remodel the ECM (overexpression and cell surface shedding) are compensated for by increases in MT1-MMP, and MMP-2 levels as seen by zymography.

Background
Remodelling of the extracellular matrix (ECM) primarily occurs through the combined action of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), and reversion-inducing cysteine-rich protein with Kazal motifs (RECK) [1]. The RECK gene encodes a glycosylphosphatidylinositol (GPI)-anchored protein with three protease inhibitor-like domains called Kazal motifs. RECK was initially classified as a tumour suppressor protein due to its ability to suppress metastasis when ectopically expressed in transformed cells [2]. In vitro studies have shown that RECK can inhibit MMP-2 and -9, secreted gelatinases associated with important developmental events and diseases, as well as MT1-MMP, a multi-functional cell surface MMP [2][3][4]. Many studies have investigated the embryonic role of MMPs. Perhaps due to redundant functions between family members, mice knockouts of each MMP, apart from MT1-MMP, and all TIMPs are viable [5]. This positions MT1-MMP as a uniquely important molecule, and therefore its regulation by other agents, such as RECK, is of interest. Likewise, studies have implicated the importance of RECK during development, as RECK knockout is embryonic lethal. In both mice and fish, loss or reduction of RECK causes defects in angiogenesis and neurogenesis, in part due to aberrant regulation of MMPs [4,[6][7][8]. In addition, RECK has been shown to play a role in cell migration signalling pathways independent of its MMP-inhibitory role [9].
Our previous studies using epithelial breast cancer cells have revealed a complex pattern of expression between MT1-MMP levels and ERK activation into pERK, as well as pro-MMP-2 activation into MMP-2. Mammalian MCF-7 cells stably expressing extremely high levels of MT1-MMP demonstrated low ERK activation and high pro-MMP-2 activation. Conversely, cells with low levels of MT1-MMP demonstrated high ERK activation and low pro-MMP-2 activation [10]. Treatment with the broad spectrum MMP inhibitor BB94 increased MT1-MMP expression, but decreased MMP-2 activation and did not alter pERK protein levels [11]. In mammalian MDA-MB-231 cells, inhibition of pro-MT1-MMP activation elevated global MT1-MMP and pERK levels, but decreased MMP-2 levels [12]. These mammalian cell studies demonstrated that when MT1-MMP levels are altered, pERK and MMP-2 activation levels alter in different ways depending on treatment. This suggests a mechanism that may involve more molecules than just MT1-MMP in the activation of ERK and pro-MMP-2, with RECK possibly playing an important role. We have previously cloned and characterized the RECK gene and studied its expression during Xenopus laevis development, showing that RECK is present at stages where ECM remodeling events are associated with neural function [13]. Our recent in vivo examination of RECK, MT1-MMP, and TIMP-2 show that these proteins colocalize in the dorsal axis of 48-h Xenopus tailbud stage embryos, particularly in the neural tube [14]. Several other studies have also described interactions between RECK and MT1-MMP proteins in vivo and in vitro [15][16][17], with RECK being shown to complex with MT1-MMP at the cell surface to both attenuate its proteolytic activity and modulate its endocytosis from the cell surface [18]. To corroborate our in vitro mammalian work that link MT1-MMP and pERK levels, as well as build on our Xenopus in vivo localization of MT1-MMP and RECK, we here used an in vitro examination of Xenopus A6 epithelial cells to confirm the importance of RECK as it relates to MT1-MMP, pERK, and MMP-2 protein levels, across poikilotherm and ectotherm species, and between in vivo and in vitro models. In this study, we used a Morpholino approach to knock down RECK levels, plasmid transfection to overexpress RECK, and PI-PLC treatment to shed RECK from the surface of A6 cells. RECK reduction did not alter MT1-MMP protein levels, ERK activation, or MMP-2 activity levels. RECK overexpression and PI-PLC treatment both resulted in increased MT1-MMP protein levels and MMP-2 activity levels. Only RECK overexpression decreased pERK protein levels in A6 cells. From these results, it is suggested that optimal levels of RECK present on the cell surface are important for modulating MT1-MMP protein levels and MMP-2 activation.

Morpholino design
The design and synthesis of Morpholinos (MO) were performed by Gene Tools (Philomath, USA). A translationblocking MO (antisense: CAT CAC ATC CCC ACT CCT TCT CTT C) was engineered to target X. laevis RECK (GenBank, AIZ00509.1). Standard scrambled MOs (antisense: CCT CTT ACC TCA GTT ACA ATT TAT A) and a carboxyfluoresceinated-labelled MO targeted to the X. laevis β-catenin gene (antisense: TTT CAA CCG TTT CCA AAG AAC CAG G) were also purchased from Gene Tools as controls.
For RECK knockdown, cells were seeded on 35 mm dishes at a density of 1 × 10 6 cells. After 24 h, spent culture medium was replaced with medium containing Endo-Porter reagent and MO oligos (Gene Tools, Philomath, USA) according to manufacturer's instructions. Forty-eight hours following treatment, cell lysates were collected. MOs were used in dosages of 1, 10, or 20 µM. Following confirmation of RECK protein decrease, 20 µM MO treatment was used in subsequent experiments.
For RECK overexpression, cells were seeded as above. Twenty-four hours later, cells were transfected with HAtagged RECK in pcDNA3.1 using Lipofectamine 2000 (Thermo Fisher, Mississauga, Canada) according to manufacturer's instructions. Another 24 h following transfection, cell lysates were collected. The generation of the full-length RECK cDNA construct is described in [13], with the HA tag being inserted just following the N-terminal signal sequence such that it would not be removed during secretion, nor would it interfere with GPI anchor formation at the C-terminal end.
Phosphatidylinositol-specific phospholipase C (PI-PLC) is an enzyme from Bacillus cereus that cleaves GPI-linked proteins, such as RECK, from the plasma membrane. Twenty-four hours following transfection, RECK-transfected and mock-transfected cells were treated with 100 U mL −1 of PI-PLC (Thermo Fisher, Mississauga, Canada) in serum-free L-15 media according to manufacturer's instructions. Twenty-four hours later, cell lysates were collected.

Quantitative real-time PCR
To investigate changes in transcript levels, real-time qPCR was performed. Cells were seeded and treated  [20].
Signal was detected using a SuperSignal Chemiluminescent kit (Thermo Scientific, Mississauga, Canada) as per the manufacturer's instructions. Blots were visualized using the Bio-Rad ChemiDoc Imaging system and quantified using Bio-Rad QuantityOne 1-D image analysis software (Bio-Rad, Mississauga, Canada). ERK1/2 activation is presented as a ratio between pERK and total ERK1/2 band intensities.

Gelatin zymography
To measure the activity of secreted MMP-2 and MMP-9 proteins, gelatin zymography was performed. Cells were seeded and treated as described above. Twenty-four hours later, culture media was replaced with serumfree L-15 media. Twenty-four hours later, serum-free media was collected and mixed with 2× SDS-loading dye (0.5 M Tris-HCl pH 6.8, 10% SDS, 2.5% glycerol, and 1% Bromophenol Blue) in a 1:1 ratio, with only 5 μL of sample being loaded into the gel [21]. To aid in the identification of active versus inactive forms of an MMP, the broad spectrum MMP inhibitor BB94 (10 μM) was added to duplicate samples. Media samples were run onan SDS-containing 15% polyacrylamide gel, co-polymerized with 1% gelatin, at 140 V for 6 h. A Triton X-100 (2.5%) incubation was then used to renature MMP-2 and MMP-9 proteins. Gels were placed in developing buffer (pH 7.5, 50 mM Tris, 200 mM NaCl, 5 mM CaCl 2 , and 0.02% Brij-35) at 37 °C with gentle shaking. After 48 h, gels were stained with 0.5% Coomassie blue for 1 h followed by destaining (50% methanol) for 2 h. Gels were visualized and band intensities representative of enzyme activity quantified using Bio-Rad (Mississauga, Canada) ChemiDoc Imaging system and QuantityOne 1-D image analysis software. As protein used for zymography was isolated from the media, the use of an internal (loading) standard was not possible. Accordingly, the ratio of active over total (pro, intermediate, and active) forms was determined for MMP-2. These ratios (active MMP-2/ total MMP-2) were then used to calculate the percentage change of MMP-2 activation levels between control and treated cells.

Statistical analysis
Statistical analysis and graphing were performed using Microsoft Excel. Data is presented as mean ± SD, except for qPCR results in Fig. 7 which are ± SEM. One-way ANOVA followed by unpaired student's t-tests were used and indicated respectively in each figure legend. The different levels of significance are denoted as follows: ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001;****, p ≤ 0.0001.

RECK reduction in A6 cells did not alter MT1-MMP or pERK protein levels nor MMP-2 activity levels
To confirm that the RECK MO treatment decreased RECK protein level in Xenopus A6 cells RECK protein was quantified following treatment with different concentrations of RECK MO. Immunoblotting demonstrated that RECK protein is endogenously expressed in untreated A6 cells. A dose-dependent decrease in cellular RECK protein level was then seen following treatment of increasing concentrations of RECK MO (Fig. 1a). The decrease in RECK was significant when immunological signals were quantified, with the 20 µM treatment reducing RECK levels by 60% (Fig. 1b). RECK level did not change following treatment of scrambled MO or β-catenin MO [unpublished observations]. Having confirmed the efficacy of the RECK MO, we examined the levels of MMPs and their activity in A6 cells following RECK knockdown. RECK reduction did not alter MT1-MMP nor pERK protein levels (Fig. 2a, b) as seen by immunoblot analysis. However, this 60% reduction in RECK was large enough to cause a cellular effect, as there was a change in transcription levels (see also Fig. 7a). Zymography was then used to quantify the active and inactive secreted forms of MMP-2 and MMP-9 after RECK reduction. Bands of gelatin degradation representative of MMP activity of specific molecular mass on zymograms were quantified. The addition of the broad spectrum MMP-inhibitor BB94 was used to confirm the identity of active versus pro-MMP forms [unpublished observations]. The amount of the active form of an MMP was compared to the total amount of MMP (pro, intermediate, and active forms). The active/total MMP ratio was set to 1 in control cells. The relative level of MMP-2 activity did not change (there was the same ratio of active to total MMP-2) after RECK reduction (Fig. 2c). MMP-9 activity could not be detected in the media (Fig. 2c) at the previously described molecular mass [10,22,23].

RECK overexpression in A6 cells caused an increase in MT1-MMP protein levels and relative active MMP-2 levels, and a decrease in pERK protein levels
After observing no change in MT1-MMP, pERK, and MMP-2 activation following RECK reduction, we proceeded with overexpression of RECK in A6 cells. Transfection of an HA-tagged RECK construct was confirmed, as the level of RECK protein in transfected cells significantly increased. This increase in protein was detected and quantified using both a RECK and HA antibody (Fig. 3a, b).
As was done with the knockdown treatment, cellular proteins of interest were quantified following RECK overexpression. Although no change was observed following RECK reduction, RECK overexpression resulted in a significant increase in the level of MT1-MMP protein (Fig. 4a) as well as a significant decrease in the level of pERK protein (Fig. 4b). Following RECK overexpression, zymography of secreted proteins revealed a significant increase in the relative level of active MMP-2 (Fig. 4c). Secreted MMP-9 proteins could not be detected via zymography (Fig. 4c).

Treatment with PI-PLC reduced cell-surface RECK levels
PI-PLC treatment (which releases GPI-linked proteins) was used to shed RECK from the cell surface, and also allowed us to confirm the proper trafficking and localization of overexpressed RECK protein to the cell surface in the A6 cells. As expected, PI-PLC treatment significantly reduced the level of membrane localized RECK, both in control and RECK-overexpressing cells (Fig. 5a, b). . β-actin is used as a loading control. b Densitometry quantification of pERK normalized to total ERK levels. pERK protein levels did not significantly change in RECK reduced cells compared to control (set to 1). c Gelatin zymography was used to measure protein activity of secreted pro-, intermediate, and active MMP-2 following RECK knockdown in A6 cells. Data is presented as the ratio between active and total (pro-, intermediate, and active) MMP-2 levels between control and RECK reduced cells. Active/total MMP-2 levels did not significantly change in the media of RECK reduced cells compared to control (set to 1). Secreted MMP-9 protein could not be detected at the expected size of 92 kDa (shown by the arrow). Graphed data is based on 3 biological replicates (mean ± SD). Data is analyzed via t-test; ns, p > 0.05 Fig. 3 Transfection of full-length HA-tagged RECK constructs in A6 cells resulted in increased RECK levels. a Immunoblot analysis was used to confirm overexpression of RECK protein following transfection of RECK cDNA constructs. Mock-transfected and GFP-transfected A6 cells were used as controls. Protein was extracted from cells 24 h following transfection. β-actin is used as a loading control. Left panel demonstrates increased levels of RECK protein compared to control using a RECK antibody, while right panel confirms the presence of HA-tagged proteins only in RECK-transfected A6 cells using an HA antibody. b Graphed data is based on 3 biological replicates (mean ± SD) and normalized to control (set to 100%). Data is analyzed via t-test; ***, p ≤ 0.001

PI-PLC treatment of A6 cells caused an increase in MT1-MMPand relative active MMP-2 levels
To elucidate the role of RECK at the cell surface in modulating MT1-MMP, pERK, and MMP-2 activation, we shed GPI-anchored proteins, which includes RECK, from the surface using PI-PLC. PI-PLC treatment significantly increased MT1-MMP protein levels ( Fig. 6a) but did not alter pERK levels (Fig. 6b). PI-PLC treatment caused an increase in the relative level of active MMP-2 (Fig. 6c). Secreted MMP-9  1). b Densitometry quantification of pERK normalized to total ERK levels. pERK protein levels significantly decreased in RECK-overexpressing cells compared to control (set to 1). c Gelatin zymography was used to measure protein levels of secreted pro-, intermediate, and active MMP-2 following RECK overexpression in A6 cells. Data is presented as the ratio between active and total (pro-, intermediate, and active) MMP-2 levels between control and RECK-overexpressing cells. Active/total MMP-2 levels significantly increased in the media of RECK-overexpressing cells compared to control (set to 1). Secreted MMP-9 protein could not be detected at the expected size of 92 kDa (shown by the arrow). Graphed data is based on 3 biological replicates (mean ± SD). Data is analyzed via t-test; *, p ≤ 0.05; **, p ≤ 0.01 protein could not be detected (Fig. 6c). Of note-PI-PLC treatment did not alter the transcription of a variety of genes (Fig. 7c), nor did it alter the morphology, growth, or survival of these cells [unpublished observations].

Alterations in RECK levels or PI-PLC treatment had varying effects on MMP-2, MMP-9, MT1-MMP, and TIMP-2 mRNA levels
Having shown that altered RECK levels had varying effects on protein levels, we sought to better understand the mechanism of these changes by examining changes in RNA levels. Following RECK reduction, qPCR analysis did not reveal a significant change in the level of MMP-2, MMP-9, nor MT1-MMP mRNA. There was, however, a significant decrease in TIMP-2 mRNA levels (Fig. 7a). Following RECK overexpression, PCR analysis revealed significant increases in MT1-MMP and MMP-9 transcript levels, but no changes in MMP-2 nor TIMP-2 transcript levels (Fig. 7b).
Of note, unlike MMP-2, MMP-9 transcript levels in A6 cells were extremely low (only being detectable after 32 PCR cycles) [unpublished observations]. Although MMP-9 transcript levels altered under RECK manipulation (Fig. 7b), we do not believe they elevated enough to be biologically relevant. Indeed, we could not detect MMP-9 proteins secreted by A6 cells via gelatin zymography even when transcript levels increased (Figs. 2c, 4c and 6c).

Discussion
Adult epithelial cells, unlike their embryonic counterparts, move and invade only under special situations, such as injury or disease. Our in vitro examination of MCF-7 epithelial breast cancer cells, which are poorly invasive, has suggested that MT1-MMP orchestrates this transition towards a more migratory phenotype [9,10,24]. While the roles of ECM remodeling proteins have garnered much interest due to their broad developmental and disease implications, it has become clear that there is not a defined relationship between the level of an MMP and the amount of ECM turnover [10]. While not exhibiting overt abilities to remodel the ECM or to become mesenchymal, non-migrating epithelial cells express low levels of the enzymes and signaling molecules needed for these events. Indeed, A6 cells have basal levels of MT1-MMP, MMP-2, RECK and pERK proteins (Figs. 1, 2). It is this low and appropriately localized levels of MT1-MMP and TIMP-2 which can directly regulate enzymatic activity, including the activation of proMMP-2 [11,25], as well as ERK cell signaling events [10,11] in mammalian epithelial cells. To address if the maintenance of low levels of MMPs is a common mechanism seen in non-mammalian epithelial cells, here we investigate the impact of altered RECK levels in the context of the ECM remodelers MT1-MMP and MMP-2, as well as ERK in Xenopus A6 cells.
The presence of MT1-MMP and active MMP-2 (measured by zymography) demonstrate that A6 cells have at least a basal ability to remodel the ECM. When the endogenous levels of RECK are reduced using MO treatment, no changes are seen in MT1-MMP and pERK protein levels, nor MMP-2 activation (Fig. 2). Despite its ability to act as an MMP inhibitor, reduced levels of RECK were not substantial enough to alter MT1-MMP or MMP-2 levels, nor the basal level of ECM remodelling that may be associated with them. However, this level RECK reduction was biologically relevant as it was enough to decrease TIMP-2 mRNA levels significantly (Fig. 7). Thus, it appears that A6 cells maintain low levels of RECK at the cell surface, levels which when further reduced, do not impact the protein levels of MT1-MMP, MMP-2, or pERK.
As RECK reduction did not alter MT1-MMP, pERK, nor MMP-2 activity levels, we next examined the effect of RECK overexpression in A6 cells. RECK impedes MT1-MMP directly by binding to it and inhibiting its activity [4], however, RECK overexpression resulted in increased MT1-MMP protein and RNA levels ( Figs. 4 and 7), and an associated increase in MMP-2 activation (Fig. 4). While A6 cells did not lower their basal ECM remodelling abilities with respect to MT1-MMP and MMP-2 in response to lowered RECK levels, increased RECK did trigger A6 cells to alter MMP levels. This increase in MT1-MMP and MMP-2 levels could be a compensatory response with the cells increasing these MMP levels to maintain a basal ability to remodel the ECM in response to the increased levels of an inhibitor. Further, RECK is associated with increased MT1-MMP endocytosis [18]. Accordingly, increased RECK levels would reduce cellsurface MT1-MMP protein levels, triggering the cell to increase MT1-MMP production. This RECK-mediated increase in MT1-MMP endocytosis rates would also limit the ability of MT1-MMP to activate ERK, thus pERK levels are reduced under RECK overexpression conditions. In the presence of increased levels of RECK, A6 cells attempt to maintain a basal level of ECM remodelling ability by increasing MMP levels.
While A6 cells could tolerate the reduction of RECK as evident in their maintained MT1-MMP and MMP-2 levels, we next sought to examine the necessity for RECK to be membrane anchored. PI-PLC treatment was used to shed RECK and other GPI-linked proteins from the cell surface. Our PI-PLC treatment of A6 cells did not alter gene expression (Fig. 7), nor did it alter the morphology, growth, or survival of these cells [unpublished observations]. By these measures, PI-PLC treatment of A6 cells was not detrimental, while immunoblot analysis did reveal a significant decrease in cell surface RECK levels (Fig. 5).
Like RECK overexpression, PI-PLC treatment of A6 cells elevated both MT1-MMP protein and MMP-2 activation levels (Fig. 6). A possible mechanism that may explain this is that while cell surface RECK may play a role in MT1-MMP endocytosis [18], solubilized


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The First Generation of iPSC Line from a Korean Alzheimer's Disease Patient Carrying APP-V715M Mutation Exhibits a Distinct Mitochondrial Dysfunction

Alzheimer's Disease (AD) is a progressive neurodegenerative disease, which is pathologically defined by the accumulation of amyloid plaques and hyper-phosphorylated tau aggregates in the brain. Mitochondrial dysfunction is also a prominent feature in AD, and the extracellular Aβ and phosphorylated tau result in the impaired mitochondrial dynamics. In this study, we generated an induced pluripotent stem cell (iPSC) line from an AD patient with amyloid precursor protein (APP) mutation (Val715Met; APP-V715M) for the first time. We demonstrated that both extracellular and intracellular levels of Aβ were dramatically increased in the APP-V715M iPSC-derived neurons. Furthermore, the APP-V715M iPSC-derived neurons exhibited high expression levels of phosphorylated tau (AT8), which was also detected in the soma and neurites by immunocytochemistry. We next investigated mitochondrial dynamics in the iPSC-derived neurons using Mito-tracker, which showed a significant decrease of anterograde and retrograde velocity in the APP-V715M iPSC-derived neurons. We also found that as the Aβ and tau pathology accumulates, fusion-related protein Mfn1 was decreased, whereas fission-related protein DRP1 was increased in the APP-V715M iPSC-derived neurons, compared with the control group. Taken together, we established the first iPSC line derived from an AD patient carrying APP-V715M mutation and showed that this iPSC-derived neurons exhibited typical AD pathological features, including a distinct mitochondrial dysfunction.

INTRODUCTION
Alzheimer' s Disease (AD) is a progressive neurodegenerative disease, and AD patients exhibit loss of memory that impairs their ability to learn or carry out daily tasks. Pathologically, AD can be characterized by the accumulation of amyloid plaques and hyper-phosphorylated tau aggregates which causes neuronal loss in the brain [1,2]. AD can be divided into sporadic and autosomal-dominant familial forms. The latter form is rare, but very tragic, given its 100% penetrance to the family members who have the causative mutations. The autosomal-dominant form of AD is associated with mutations in amyloid precursor protein (APP), presenilin-1 (PS1), or presenilin-2 (PS2) [3]. It has been demonstrated that the APP C-terminal mutations including APP (V715M) exhibited increased Aβ 42 secretion in primary neurons [4,5]. The APP (Val715Met; APP-V715M) mutation was detected in Korea and the patient showed typical clinical symptoms [6]. In this study, we generated an induced pluripotent stem cell (iPSC) line from an AD patient with APP-V715M mutation for the first time. We characterized the phenotypes of APP-V715M iPSC-derived neurons, including extracellular and intracellular levels of Aβ and high expression levels of phosphorylated tau, compared with the elderly normal control iPSC line which has been fully characterized in our previous study [7]. We investigated the mitochondrial dynamics and the expression of fission and fusion-related proteins in the iPSC-derived neurons. APP-V715M iPSC-derived neurons exhibited decrease of mitochondrial velocity and impaired balance of mitochondrial fission and fusion, indicative of extracellular Aβ or phosphorylated tau-induced impairment of mitochondrial dynamics [8][9][10][11]. Taken together, we established an iPSC line, for the first time, from a middle-aged AD patient with an APP-V715M mutation, which recapitulats the cardinal features of AD pathophysiology, which will be potentially useful to develop biomarkers associated with disease progression and responses to the diseasemodifying therapies.

Enrollment of participants
The patient met the criteria for AD as recommended by the National Institute on Aging-Alzheimer's Association, and the normal elderly subject fulfilled the criteria of a normal elderly control as defined by Christensen, et al [12,13]. We performed detailed neuropsychological tests, MRI, [ 18 F]-Florbetaben amyloid PET, and blood draw for iPSC generation from each participant. The institutional review board of the Samsung Medical Center, Asan Medical Center, and CHA University approved the study protocol, and informed written consent was obtained from each participant.

Characterization and differentiation of iPSCs
Mononuclear cells (MNCs) were isolated freshly from the peripheral blood of the APP-V715M patient using the Ficoll-Paque TM PLUS method (GE Healthcare, USA) [7]. Isolated peripheral-derived MNCs (PBMCs) were infected with SeVdp (KOSM) 302L [14]. Genotyping of the APP-V715M single nucleotide mutation was performed by DNA sequencing (Cosmo Genetech, Korea). The APP gene was amplified by PCR using the following primers (forward primer: TTC AAG GTG TTC TTT GCA GA; reverse primer: CAT AGT CTT AAT TCC CAC TTG G). For teratoma formation, undifferentiated iPSCs were harvested and subcutaneously transplanted into NOG mice. Teratomas were dissected and fixed with 4% paraformaldehyde (PFA) at 8 to 16 weeks after injection. Paraffin-embedded tissue was sectioned and stained with hematoxylin/eosin to detect the formation of threegerm layer tissue morphology. Karyotyping, Sendai virus detection PCR, in vitro and cortical differentiation were performed as we described before [7,14].

Extracellular and intracellular amyloid-β ELISA
Extracellular Aβ levels were measured using conditioned media (CM), which were collected from cultured neuronal cells (1×10 5 ) at 48 hours after the last medium change from 8 and 10 weeks of differentiation. Intracellular Aβ 42 and Aβ 40 were measured in a total of 1µg proteins from 10 week-differentiated neurons. All procedures were essentially same as described before [7].

Immunocytochemistry and Western blot
Immunocytochemistry and Western blot analysis were performed as described before [7]. The following primary antibodies were used: anti-OCT4

Live cell imaging and mitochondrial dynamics analysis
Living cells were imaged using Leica TCSSP5II confocal microscopy. Ten week-differentiated neurons were incubated with Mitotracker red (Thermo-Fisher Cat. imaging (LCI) analysis. Cells were maintained at 37℃ and were supplied with atmosphere of 5% CO 2 /95% air (Live Cell Instrument, Seoul, Korea) during imaging. Time-lapse image recording were acquired in 2 sec interval and duration up to 4 min 30 sec. Mitochondria kymographs were analyzed using KymographClear, an ImageJ macro toolset that allows for the generation of kymographs from image sequences. Quantitative analysis of mitochondria velocity was performed using KymographDirect, a standalone tool to extract quantitative information from kymographs in an automated way with high accuracy and reliability [15].

Statistical analysis
All statistical analyses were performed using the Student' s t test or one-way analysis of variance (ANOVA) following the Fisher' s LSD (Least Significant Difference) in the Statistical Analysis System (Enterprise 4.1, SAS Korea, Seoul, Korea). Significance was accepted at the 95% probability level. Data in graphs were presented as mean ± SEM. Statistical significance (i.e., p value) in the graphs were presented as follows: p<0.05 (*), p<0.01 (**) and p<0.001 (***).

Clinical features of the patient
This study is based on a 54 year-old man with an APP muta-tion (Exon17; c.2143G>A; p.V715M) who visited the memory disorder clinic of Asan Medical Center (Seoul, Korea). The patient developed symptoms at the age of 49, starting with memory impairment and attention deficits. He subsequently developed an impairment in daily activities at the age of 52. Neurological examination revealed a generalized cognitive impairment with the mini-mental state examination (MMSE) score of 10. Treatments with cholinesterase inhibitor and NMDA receptor antagonist had limited effects. By the age of 54, the patient developed bradykinesia and rigidity, and no longer understood verbal commands. MRI taken at the age of 54 showed a generalized cortical atrophy, and the 18 F-Florbetaben amyloid PET showed a significant amyloid accumulation in the bilateral striatum and association cortices (Fig.  1A). The patient had a strong family history of dementia with an autosomal dominant pattern (Fig. 1B). A 72 year-old healthy man with normal cognition was enrolled for the study. He showed no brain atrophy on brain MRI and no significant amyloid accumulation on amyloid PET (Fig. 1A). Informed written consent was obtained from each participant.
To investigate the pathological features of the patient with APP-V715M mutation, we generated an iPSC line from an AD patient carrying the APP-V715M mutation using the technology that we developed for iPSC generation [7]. The APP patient-derived MNCs were reprogrammed using Sendai virus vector (SeVdp) which expresses four reprogramming factors (OCT3/4, SOX2, cMYC, and KLF4) [7,14]. For iPSC generation, we picked more than three individual clones and selected the best growing clone for further analyses. iPSC lines harboring the APP-V715M mutation exhibited the typical expression of pluripotent stem cell markers, including OCT4, SOX2, SSEA4 and TRA-1-81 ( Fig. 2A). Differentiation potential of iPSC lines was assessed using in vitro embryoid body formation and in vivo teratoma assays, based on the three-germ layer marker expression ( Fig. 2B and 2F). Genotyping of the established iPSC line was confirmed by a conventional sequencing method (Fig. 2C). No SeVdp vector was integrated in the established iPSC line (Fig. 2D) and its karyotype was normal (Fig. 2E).
Although the effects of overexpression of the APP-V715M mutation on Aβ secretion have been studied in primary neurons previously [4,5], little has been known about the pathological features of the patients carrying the APP-V715M mutation at cellular levels. To investigate the Aβ levels in the APP-V715M iPSCderived neurons, we measured Aβ 40 and Aβ 42 levels both extracellularly and intracellularly at 10 weeks of neuronal differentiation.
No significant difference in Aβ 40 level was detected. However, the APP-V715M iPSC-derived neurons exhibited a dramatic increase in Aβ 42 level (i.e., over 2-fold) at 10 weeks of differentiation. Importantly, the ratio of Aβ 42 /Aβ 40 was significantly increased (i.e., over 2-fold) in the APP-V715M iPSC-derived group, compared with the control group (Fig. 3A). To detect the Aβ deposits, the iPSC-derived neurons were stained with anti-Aβ 42 antibody at 10 weeks of neural differentiation. Notably, confocal microscopy images showed a robust increase in the extracellular Aβ 42 deposits in the APP-V715M iPSC-derived neurons, compared to the control (Fig. 3B). Furthermore, we extracted the TBS-insoluble/ SDS-soluble fraction according to the previous protocols [16,17]. The TBS-insoluble/SDS-soluble intracellular Aβ 42 and Aβ 40 levels were measured in a total 1µg of proteins using ELISA at 10 weeks of neuronal differentiation. Levels of the intracellular Aβ 42 and the ratio of Aβ 42 /Aβ 40 were significantly increased in the APP-V715M iPSC-derived neurons (Fig. 3C). Western blot analysis showed that the expression level of total APP was significantly decreased in the APP-V715M iPSC-derived neurons ( Fig. 3D and 3E). By contrast, the Aβ oligomers showed a significant increase in the APP-V715M iPSC-derived neurons, compared with the control (Fig. 3D and  3F). It has been recently demonstrated in vitro that the increase and accumulation of Aβ peptides can lead to the formation of neurofibrillary tangles consisting of aggregated hyperphosphorylated tau [16]. Tau protein normally exists as a soluble form in the axons of mature neurons. However, when it becomes hyperphosphorylated, p-tau protein abnormally accumulates in the dendrites and the cell bodies in AD [18,19]. To investigate the p-tau levels in the APP-V715M iPSC-derived neurons, we performed an immunocytochemical analysis using an antibody against AT8 (phosphorylated at Ser202/Thr205). We found that the APP-V715M iPSCderived neurons exhibited a significant increase of AT8 expression in the neurites and the soma, compared with the control group (Fig.  3G~I). Furthermore, we performed Western blot to measure the AT8 protein levels and found that the APP-V715M iPSC-derived neurons showed an increase of tau levels, compared with the control group (Fig. 3J~L). Taken together, we demonstrated that the APP-V715M iPSC-derived neurons exhibited significant increases both in extracellular and intracellular Aβ levels and increased levels of p-tau levels, compared with the control group.
To investigate the mitochondrial dynamics, we performed the live cell imaging analysis using Mito-tracker (Ds-Red) at 10 weeks of neuronal differentiation. We found that the APP-V715M iPSCderived neurons showed a reduced movement compared with the control group (Supplementary movie files 1 and 2). Kymograph (Fig. 4A) and the quantification analysis of mitochondria velocity were performed using KymographClear and KymographDirect, respectively [15]. Both anterograde and retrograde velocity were significantly decreased in the APP-V715M iPSC-derived neurons compared with the control group ( Fig. 4B and 4C). Furthermore, we investigated the expression levels of mitochondrial fusion and fission-related proteins at 6 and 10 weeks of neuronal differentiation, including mitochondria fusion-related protein Mfn1 (membrane proteins mitofusin 1), Mfn2 (membrane proteins mitofusin 2) and mitochondria fission-related proteins DRP1 (dynaminrelated protein 1) and Fis1 (mitochondrial fission 1 protein). There was no significant difference of fusion and fission-related protein expression in the control and APP-V715M iPSC-derived neurons at 6 weeks of neuronal differentiation. At 10 weeks of neuronal dif-ferentiation, Western blot analysis revealed that Mfn1 expression was dramatically reduced, whereas DRP1 expression levels were significantly increased in the APP-V715M iPSC-derived neurons, compared with the control neurons ( Fig. 4D and 4E). In accordance with previous reports, in which accumulated Aβ and p-tau affect mitochondrial functions, our results strongly suggest that high levels of Aβ and p-tau may disrupt the mitochondrial transport, presumably due to the impaired balance of mitochondrial fusion and fission in the APP-V715M iPSC-derived neurons.
Mitochondria plays an important role in neuronal function and survival, and mitochondrial dysfunction is one of the most prominent features in the AD brains [9]. We investigated the mitochondrial dynamics in the APP-V715M iPSC-derived neurons, which exhibited a dramatic decrease of mitochondria movement as well as the anterograde and retrograde mitochondrial velocity using Mito-tracker (Supplementary movie files 1, 2 and Fig. 4). Mitochondrial dynamics is determined by the constantly repeating process of fusion and fission, and this process is tightly regulated by the balance of fusion and fission-related proteins [10,20]. As extracellular Aβ and phosphorylated tau are implicated in the impairment of mitochondrial fission and fusion processes in humans [10,20,21], we measured the expression level of fusion and fissionrelated markers at 6 and 10 weeks of neuronal differentiation, because APP-V715M iPSC-derived neurons showed a significant increase in Aβ levels from 8 weeks of neuronal differentiation (Fig.   Fig. 3 Mitochondrial Dysfunction in AD-iPSC (APP-V715M) 3A). Western blot analysis revealed that the expression of fissionassociated markers was significantly increased, but fusion-related markers were significantly decreased in the APP-V715M iPSC-derived neurons at 10 weeks. However, this difference was not clearly detected at 6 weeks of neuronal differentiation ( Fig. 3D and 3E). Based on these findings, we speculated that the increased Aβ secretion and phosphorylated tau proteins can lead to the defective mitochondrial axonal transport and the imbalance of mitochondrial fission and fusion in the APP-V715M iPSC-derived neurons.
In summary, we generated an iPSC line from AD patient with the APP-V715M mutation for the first time. We also characterized the pathological features of APP-V715M iPSC-derived neurons, including increased levels of extracellular and intracellular Aβ as well as phosphorylated tau, and identified that mitochondrial dysfunction may be a key contributing factor to AD pathophysiology in the APP-V715M iPSC-derived neurons.