Alzheimer’s disease associated AKAP9 I2558M mutation alters posttranslational modification and interactome of tau and cellular functions in CRISPR‐edited human neuronal cells

Abstract Alzheimer's disease (AD) is a pervasive neurodegeneration disease with high heritability. In this study, we employed CRISPR‐Cas9‐engineered technology to investigate the effects of a rare mutation (rs144662445) in the A kinase anchoring protein 9 (AKAP9) gene, which is associated with AD in African Americans (AA), on tau pathology and the tau interactome in SH‐SY5Y P301L neuron‐like cells. The mutation significantly increased the level of phosphorylated tau, specifically at the site Ser396/Ser404. Moreover, analyses of the tau interactome measured by affinity purification‐mass spectrometry revealed that differentially expressed tau‐interacting proteins in AKAP9 mutant cells were associated with RNA translation, RNA localization and oxidative activity, recapitulating the tau interactome signature previously reported with human AD brain samples. Importantly, these results were further validated by functional studies showing a significant reduction in protein synthesis activity and excessive oxidative stress in AKAP9 mutant compared with wild type cells in a tau‐dependent manner, which are mirrored with pathological phenotype frequently seen in AD. Our results demonstrated specific effects of rs14462445 on mis‐processing of tau and suggest a potential role of AKAP9 in AD pathogenesis.


| INTRODUC TI ON
Alzheimer's disease (AD) is the most common form of dementia with a high genetic influence. A growing number of common genetic variants associated with AD have been identified by genomewide association studies, although with the exception of APOE ε2 and ε4 alleles, their impact on disease risk are very modest (Kunkle et al., 2019). Next-generation sequencing has identified rare variants in several genes that exert moderate to high effects on disease risk compared with most common variants (Guerreiro et al., 2013).
Indeed, our previous whole exome sequencing study identified two rare African American (AA)-specific variants in AKAP9, a kinase anchor protein 9 (rs144662445 and rs149979685) that increased the odds of AD more than 2.75-fold (Logue et al., 2014). AKAP9 is a binding protein that tethers protein kinase (PKA) to intracellular location to increase its catalyzing abilities by enhancing its sensitivity to cAMP (Terrin et al., 2012). It can recruit adenylyl cyclase to generate cAMP required for PKA activation (Piggott et al., 2008). It has been reported that PKA enhances tau phosphorylation by promoting glycogen synthase kinase-3β (GSK-3β) activity (Liu et al., 2006). Activation of PKA, leading to hyperphosphorylated tau which is one of the pathological hallmarks of AD, was found in brain tissue from neuropathologically confirmed AD cases (Duka et al., 2013). Interestingly, rs144662445 (I2558 M) is located within the PKA regulatory subunits binding site of the AKAP9 Q99996-1 transcript (Logue et al., 2014;Terrin et al., 2012), suggesting that this mutation may alter the activation status of PKA and thus alter the phosphorylation level of tau protein. This idea is supported by our previous findings showing that rs144662445 increases tau phosphorylation without affecting amyloidβ in lymphoblastoid cell lines (Ikezu et al., 2018;McCahill et al., 2005). However, the functional impact of the AKAP9 I2558 M mutation in the central nervous system (CNS), and how it might contribute to AD-related tauopathy AD remain unclear.
SH-SY5Y human neuroblastoma cells are CNS-derived, easily cultured and known to display tau phosphorylation innately with some similarities to AD in human brain (Kovalevich & Langford, 2013;Tanaka et al., 1995;Zhong et al., 1999). They can be differentiated to present a neuronal morphology and phenotype mimicking primary neurons (Agholme et al., 2010;Greene et al., 2020). SH-SY5Y cells that express the P301L human tau mutation constitutively provide a useful and relevant AD model for in vitro experiments (de Medeiros et al., 2019;Mirra et al., 1999).
Using CRISPR-Cas9 technology, we successfully tested the potential effects of the AKAP9 I2558 M mutation on tau pathology in SH-SY5Y cells stably expressing P301L tau and analyzed the tau interactome using data generated by affinity-purified Mass spectrometry. We found increased expression of phosphorylated tau in cells with the I2558 M mutation, specifically at the site Ser396/Ser404. Further, our studies revealed that I2558 M leads to significant differences in tau-interacting proteins and recapitulates phenotypes observed in AD, such as dysregulation of protein synthesis and excessive oxidative stress.

| CRISPR-Cas9-mediated AKAP9 I2558 M mutation knock-in
SH-SY5Y P301L cells with the AKAP9 I2558 M mutation were generated using CRISPR gene editing technology. Based on the genomic sequence of human AKAP9 (Genbank ID 10142), gRNA was designed with the sequence "ACCAGAGAATAGTGTTAACG" targeting the area near the mutation site using the CRISPR design tool (crispr.mit. edu). Cleavage efficiency was calculated to be 36.3% by sequencing trace analysis with the online tool TIDE (https://tide-calcu lator. nki.nl/). A donor template (ssODN/plasmid) was designed containing the wild type A or mutation G allele on gRNA recognition sites.
AKAP9 was targeted and mutated by transient co-transfection of plasmids carrying the gRNA, Cas9, and ssODN. The transfected cells were plated in 96-well plates by limit dilution to generate isogenic single clones. The clones were picked from wells and screened by restriction endonuclease digestion and DNA sequencing to identify isogenic modified clones. Finally, the SH-SY5Y P301L/I2558 M modified cell line was successfully generated, expanded, and tested as mycoplasma-free for subsequent experiments. Top 5 off-targets resulting from AKAP9-sgRNA T1-mediated editing were predicted by CRISPRater tool (http://crispr.cos.uni-heide lberg.de/). Three potential off-targets were validated in edited SH-SY5Y P301L cells by Sanger sequencing. The primers for PCR amplification were provided as below.
in AD. Our results demonstrated specific effects of rs14462445 on mis-processing of tau and suggest a potential role of AKAP9 in AD pathogenesis.

K E Y W O R D S
a kinase anchoring protein 9, Alzheimer's disease, CRISPR, oxidative stress, phosphorylated tau, protein synthesis, proteomics, Tau, Tau interactome

| Human tau immunoprecipitation (IP)
Human tau IP from differentiated SH-SY5Y cells (N = 5 for WT, N = 5 for I2558 M mutation, N = 3 for IgG control with a mixture of WT and I2558 M cell line samples) was processed as previously described (Ikezu et al., 2018). Briefly, cells on 10 cm dishes were washed and pelleted by centrifugation at 300 × g for 5 min at 4 10 μg of Tau 13 antibody (provided by Binder/Kanaan laboratories) (Combs et al., 2016), or normal mouse IgG antibody (Santa Cruz Biotechnology) as negative control was directly conjugated to the columns containing resins. IP samples were eluted using the elution buffer from the columns, and re-suspended in 1× Laemmli sample buffer (Bio-Rad) with sonication at 100% amplitude for eight mins, and heated at 95°C for 10 min. Cell Signaling Technology, 7074S) for 1 h, and immunoreactivity was detected using enhanced chemiluminescence solutions (Millipore, WBKLS0100). The membrane was finally visualized using the digital chemiluminescent imager (C300, Azure Biosystems). The band intensity was digitally measured using ImageJ (NIH). Scientific), the entire protein region of the gel was excised and subjected to in-gel trypsin digestion (Cat# 03708985, Roche) after reduction with dithiothreitol and alkylation with iodoacetamide overnight at 37°C as previously described (You et al., 2020). Briefly, peptides eluted from the gel were lyophilized and re-suspended in 25-50 µl of 5% acetonitrile (0.1% (v/v) FA) with 2 pmol ADH digest. A 1-2 µl injection was loaded by a Waters NanoAcquity UPLC in 5% acetonitrile (0.1% formic acid) at 4 µl/min for 4 min onto a 100 µm I.D. fused-silica pre-column packed with 2 cm of 5 µm (200 Å) Magic C18AQ (Bruker-Michrom).

| In-gel digestion and LC-MS/MS analysis
Peptides were eluted at 300 nL/min from a 75 µm I.D. gravity-pulled analytical column packed with 25 cm of 3 µm (100 Å) Magic C18AQ particles using a linear gradient from 5 to 35% of mobile phase B (acetonitrile +0.1% formic acid) and mobile phase A (water +0.1% formic acid) over 60 min. Ions were introduced by positive electrospray ionization via liquid junction at 1.4-1.6 kV into an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (ThermoFisher Scientific). Mass spectra were acquired over m/z 300-1750 at 70,000 resolution (m/z 200) with an AGC target of 1 × 10 6 , and data-dependent acquisition selected the top ten most abundant precursor ions for tandem mass spectrometry by HCD fragmentation using an isolation width of 1.6 Da, max fill time of 110 ms, and AGC target of 1e 5 . Peptides were fragmented by a normalized collisional energy of 27, and fragment spectra acquired at a resolution of 17,500 (m/z 200).

| Proteomic analysis
Raw data files were peak processed with Proteome Discoverer (version 2.1, Thermo Scientific) followed by identification using Mascot Server visualized as proportion of modified reads out of total reads. These data were then analyzed for differences in PTMs of tau protein between WT and I2558 M mutation cells using a nonparametric t-test.

| Enzyme-linked immunosorbent assay (ELISA)
Differentiated SH-SY5Y cells expressing AKAP9 WT and I2558 M mutation were lysated in RIPA buffer (Thermo Fisher Scientific) supplemented with 1% Triton-X100 and Pierce HALT inhibitor used to assess total tau, p-tau levels according to manufacturer's instructions. All measurements and data analyses were performed blind to AKAP9 genotype. We performed 6-9 replicates within each AKAP9 line from three independent experiments for the quantitation.
For tau silencing experiment, siRNA transfected cells were used for ELISA assays. 3-6 replicates within each AKAP9 line were performed for the quantitation.

| Protein synthesis assay
The detection of protein synthesis in AKAP9 WT and I2558 M muta- obtained using a live cell imaging system (IncuCyte ZOOM system, Essen BioScience) in a humidity chamber at 37°C and 5% CO2 as previously described (You et al., 2020). Images were acquired from 9 fields per culture well every 15 mins for total 1.5 hr and further The fluorescence density (O.C.U per μm 2 ) was measured by ImageJ software (NIH). Two independent experiments were performed for the comparison between the WT and I2558 M mutations.

| Statistical analysis
Graphical data were analyzed using GraphPad Prism versions 6.0.
Data are presented as mean ±SEM. Comparisons of PTMs of tau protein between two groups were analyzed by using an unpaired t-test without multiple corrections. Comparisons among two groups were analyzed using unpaired t test for normal distributed data and Mann-Whitney test for non-normal distributed data. Comparisons among groups with two factors (AKAP9 genotype and drug or siRNA treatment) were analyzed using regular two-way ANOVA with Sidak's multiple corrections.

| AKAP9 I2558 M mutation increases tau phosphorylation at Ser396/Ser404 sites
Our previous findings demonstrated that the I2558 M mutation impacts tau phosphorylation level but not amyloidβ in lymphoblastoid cell lines, particularly those from persons with AD (Ikezu et al., 2018).
To determine whether the tau phosphorylation pattern is recapitulated in neuronal cells and potentially contributes to tau pathology, we quantified tau level by the immunofluorescence using HT7 antibody for total tau, and PHF1 (detecting pSer396 and pSer404 tau;

| Distinct tau interactome and PTMs in differentiated SH-SY5Y P301L cells with AKAP9 I2558 M mutation
It is well established that post-translational modifications (PTMs) of tau including phosphorylation could alter the portfolio of its interacting proteins (Drummond et al., 2020;Trushina et al., 2019;Wesseling et al., 2020). We examined specific differences in tau-interacting proteins caused by the I2558 M mutation. As shown in Figure 3a, tau protein was immunoprecipitated by incubation with tau-13 antibody or an isotype control IgG-conjugated beads, resulting in 13 samples (AKAP9 WT: AKAP9 I2558 M: IgG = 5: 5: 3) that were digested for peptides to be individually analyzed through mass spectrometry.
Using Western blotting, we confirmed the proper immunoprecipitation of tau utilizing the tau-13 antibody, whereas no tau signal was  Table S1).
Next, we investigated post-translational modifications (PTMs) of tau protein at individual amino acids by mass spectrometry in WT and mutant cells. Nineteen different modified tau residues were identified including phosphorylation of the Ser396 and Ser404 sites (Table S2). These two residues were detected in only some co-IP PTMs of tau protein, including pyroglutamate conversion of residue Q6, oxidation of residue M11 and M31, were also significantly more frequent in I2558 M compared with WT cells (p < 0.05, Figure 4).

| AKAP9 I2558 M mutation is associated with DEPs involved in translation, RNA metabolism, and oxidative activity
Gene ontology (GO) and pathway analysis of DEPs identified in the tau interactome in WT and I2558 M mutant SH-SY5Y P301L cells showed that the tau interactome in I2558 M cells was most significantly enriched in proteins associated with translation and RNA metabolism, represented by elongation factors (e.g., EEF1G, EEF1D, EEF2, and EIF3E), ribosomal proteins (e.g., RPL4, RPL6, and RPL7) and RNA binding proteins (Figure 5a; Table S4). We confirmed the trend of increased expression by Western blotting ( Figure S4a) and cytoplasmic mislocalization of EIF3E in AKAP9 mutant cells by immunostaining ( Figure S4b). In addition, we found significant enrichment of proteins involved in oxidoreductase activity, including ubiquinone oxidoreductase subunits (NDUFA8, NDUFA10, F I G U R E 5 Functional enrichment analysis of DEPs in AKAP9 I2558 M group versus AKAP9 WT group. (a) Bar graph of the statistically enriched terms in gene ontology and reactome pathway across input proteins via the Metascape software. Total 26 DEPs in AKAP9 WT and 153 DEPs in AKAP9 I2558 M group were input. (b) Network layout of the enriched terms for DEPs of AKAP9 WT and AKAP9 I2558 M group. The size of a node is proportional to the number of input genes that fall into that term, and the respective color represents its cluster identity. Terms with a Kappa-statistical similarity score >0.3 are linked by an edge (the thickness of the edge represents the similarity score).
(c) Protein-protein interaction (PPI) network and MCODE components identified in the DEPs of AKAP9 I2558 M. MCODE algorithm was applied to PPI network to identify neighborhoods where proteins are densely connected. Each MCODE network is assigned a unique color. GO enrichment analysis was applied to each MCODE network to assign "meanings" to the network component. The red represents protein network involved in translation; the blue represents protein network involved in RNA localization; the green represents protein network involved in oxidative activity | 13 of 18 YOU et al.

NDUFS2
) in AKAP9 mutant cells. Intriguingly, a notable gene ontology termed as "misfolded protein binding" was identified with the increased expression of MAPT (tau protein) and heat shock proteins HSPA1B and HSPA4 in the tau interactome in AKAP9 mutant cells, suggesting that the I2558 M mutation may promote tau misfolding.
To further determine the role of the I2558 M mutation in AD pathology, we used the SAINT (Significance Analysis of INTeractome) algorithm to identify tau interactors in AKAP9 WT and mutant cells and compared those with proteins involved in the tau interactome in human AD brain tissue. A SAINT score representing the statistical probability of a given protein as a bona fide interactor, was determined for each protein ranging from 0 (lowest probability) to 1 (highest probability) (Teo et al., 2016). We identified significant interactions of tau with 57 proteins (SAINT score ≥0.65) in I2558 M mutant cells, including ribosomal proteins and RNA binding proteins, and with 34 proteins in WT cells, including mitochondria membrane proteins ( Figure S5). Notably, comparison of the tau interactome in I2448 M mutant cells with a human AD brain-derived tau interactome (Drummond et al., 2020) revealed 43 overlapping proteins including MAP4, PIN1, MAPT, and STXBP1, providing further evidence for an association of the I2558 M mutation with AD pathology ( Figure S6).

| AKAP9 I2558 M mutation leads to decreased protein synthesis and excessive oxidative activity in SH-SY5Y P301L neurons compared with AKAP9 WT in a tau-dependent manner.
Bioinformatics analyses of the tau interactome dataset revealed that the I2558 M mutation might affect translation and oxidation. To validate these findings, we measured protein synthesis and oxidative activity in SH-SY5Y P301L neurons with and without I2558 M using the Click-iT protein synthesis kit (Figure 6a). We found that protein However, we observed a significant higher protein synthesis activity after MAPT-siRNA transfection than that after NC-siRNA treatment in AKAP9 mutant cells, although the level was still lower compared F I G U R E 6 AKAP9 I2558 M mutation significantly inhibits the nascent protein synthesis in SH-SY5Y P301L neurons compared with AKAP9 WT in a Tau-dependent manner. (a) The scheme of protein synthesis assay. 1% DMSO or 0.01 mg/ml CHX (Cyclohexamide, protein synthesis inhibitor) were added to SH-SY5Y P301L neurons and incubated for 16 h. Evaluation of protein synthesis was performed by using Click-iT protein synthesis kit according to the manufacturer's instructions.

| DISCUSS ION
This is one of the first studies to investigate effects of an ADassociated AKAP9 mutation on AD-related pathology. We observed a higher expression of phosphorylated tau in the differentiated cells containing the I2558 M mutation that was introduced with the assistance of CRISPR-editing technology. Our results are consistent with previous observations of increased tau phosphorylation in human lymphoblastoid cell lines containing this mutation (Ikezu et al., 2018). Moreover, we obtained evidence that the I2558 M mutation promotes tau hyper-phosphorylation specifically at the S396 and S404 sites as indicated by results from ICC, ELISA, Western blotting, and tau PTMs. The 396 and 404 residues are located at the C-terminal of tau and these epitopes are associated with intracellular and extracellular filamentous tau (Cavallini et al., 2013).
Hyper-phosphorylated tau at S396/S404 is known to accelerate tau aggregation and AD-linked neurofibrillary tangle pathology (Torres et al., 2021). In addition, we observed an increased modification of pyroglutamate at Q6 of tau protein in AKAP9 mutant cells.
Pyroglutamate at Q6 has been reported to induce a cyclization at the N-terminal of tau and cause higher aggregation of Aβ plaques and conceivably tau due to increased hydrophobicity (Moro et al., 2018;Nisbet et al., 2015). Thus, our findings indicate that the deleterious effects of the I2558 M mutation may be a potential contributing factor to AD pathology.
Phosphorylation of tau protein is regulated by several kinases, including GSK-3β, CDK5, creatine kinase (CK) and PKA (Tenreiro et al., 2014), as well as phosphatases including protein phosphatase 2 (PP2A) (Sontag & Sontag, 2014). For example, exogenous expression of CK1δ increases tau phosphorylation at S396/S404, while CK1δ inhibition significantly reduces phosphorylation at this site (Lee & Leugers, 2012). Alterations in GSK-3β levels were associated with changes in tau phosphorylation including S396 in several cell and animal models (Hernandez et al., 2013). PP2A, the major tau phosphatase in human brain, was reported to dephosphorylate tau directly at several sites including Ser404, or indirectly by regulating GSK-3β activity (Qian et al., 2010). This could suggest that the I2558 M mutation may alter the interaction of these kinases or phosphatases with tau. Indeed, a previous study showed that the activity of GSK-3β on tau phosphorylation was regulated by PKA, which is known to bind AKAP9 directly (Liu et al., 2006). Our data showed that the expression of tau kinases including GSK-3β, PKA, CDK5 was not changed, while the level of PPP2CB, one of the PP2A catalytic subunits, was reduced in AKAP9 I2558 M mutation. It is possible that AKAP9 I2558 M mutation may disrupt PP2A-mediated tau de-phosphorylation, leading to hyper-phosphorylated tau level.
However, additional studies are needed to understand the details of how I2558 M may regulate the function of PP2A, and whether the activation of tau kinases is disrupted.
Our results also revealed significant differences in the tau interactome between WT and the I2558 M mutation in SH-SY5Y P301L differentiated cells. The mutant cells were enriched in tau-interacting proteins involved in translation (e.g., EEF2, RPL4, RPL6, RPL17, and RPS6). Interestingly, dysregulation of mRNA translation has been reported as a key process leading to AD pathology (Ghosh et al., 2020).

Impaired protein synthesis mediated by alterations in both ribosomal
nucleic acids and polyribosomal complexes was found in the earliest stages of AD (Ding et al., 2005). Additionally, we observed that RNA binding proteins including HNRNPU, HSPA4, EIF2S3, EIF3E, and EIF4H were among DEPs in the tau interactome in AKAP9 mutant cells. Disruptions in these proteins have been implicated in the formation of stress granules and tau pathology (Wolozin & Ivanov, 2019). Indeed, the stress granule marker EIF3E (Lee et al., 2021) has the tendency of increased expression, as well as abnormal aggregation and mislocalization in cytosol of AKAP9 mutant cells. We also found that AKAP9 mutant cells were enriched for tau-interacting proteins related to oxidative activity, including APEX1, NDUFS2, and NDUFA10. Oxidative stress has been recognized as a contributing F I G U R E 7 Elevated oxidative stress in SH-SY5Y P301L neurons with AKAP9 I2558 M mutation in a Tau-dependent manner. (a) The scheme of measuring reactive oxygen species (ROS) in SH-SY5Y P301L neurons. SH-SY5Y neurons were incubated with Vehicle (VEH) or 50 μM N-acetyl cysteine (NAC, ROS inhibitor) for 1 h before labeling with CellRox Orange Reagent. The ROS level was tracked and analyzed by using live cell tracking instrument (Incucyte). (b) Representative images of ROS signal labeled by CellRox Orange in AKAP9 WT and AKAP9 I2558 M cells. Scale bar, 200 μm. (c) Quantification of the fluorescence intensity of CellRox Orange in AKAP9 WT and AKAP9 I2558 M group. N = 2 independent experiments. All data are presented as the mean ±SEM, *p < 0.05, using two-way ANOVA with Sidak's multiple comparisons. (d) Representative images of ROS signal labeled by CellRox Green in AKAP9 WT and AKAP9 I2558 M cells with either NC-or MAPT-siRNA treatment. 10 nM NC-or MAPT-siRNA were added to day 14 SH-SY5Y P301L neurons and incubated for 6 h followed by refreshing media and culturing until day 18. The ROS level was labeled by CellRox Green reagent and fixed for analyzing by confocal microscopy. Scale bar, 20 μm. (e) Quantification of the fluorescence intensity of CellRox Orange in AKAP9 WT and AKAP9 I2558 M group. N = 2 independent experiments. All data are presented as the mean ±SEM, *p < 0.05, **p < 0.01, ***p < 0.001 using two-way ANOVA with Sidak's multiple comparisons factor to aging and progression of multiple neurodegenerative diseases including AD (Tonnies & Trushina, 2017). The increase of ROS could enhance the production and accumulation of amyloidβ and hyperphosphorylated tau protein (Cheignon et al., 2018). These data demonstrate that the I2558 M mutation may alter the tau interactome, thereby disrupting translation and RNA metabolism and inducing the oxidative stress. This conclusion was supported by functional experiments that revealed a defect in protein synthesis and excessive production of oxidative stress level which were dependent on the presence of tau in AKAP9 mutant cells.
Furthermore, the tau interactome in AKAP9 mutant cells was similar to the human phosphorylated tau interactome in AD, suggesting that AKAP9 may have an important role in AD-associated tau pathology (Drummond et al., 2020). For example, PIN1, which was significantly enriched in cells with the I2558 M mutation, also emerged as a pTau interactor and enriched in neurofibrillary tangles in AD cases (Drummond et al., 2020). A recent study showed that PIN1 dysfunction aberrantly increased tau phosphorylation and aggregation (Park et al., 2019). STXBP1, which was not enriched in I2558 M mutant cells, may regulate tau trafficking in several neurodegenerative diseases and is highly associated with pTau in AD (Lanoue et al., 2019). Additionally, there are some abundant tauinteracting proteins in AKAP9 mutation identified as either pTau interactors in AD (e.g., PSMC5 and PSMD8) or highly enriched in AD NFTs, including CCT3, CCT7, DDX1, EEF2, EZR, PSMA3, and PSMA7 (Drummond et al., 2020).
Our study has notable limitations. First, we examined the AKAP9 I2558 M mutation in differentiated SH-SY5Y cells, but future studies utilizing AD patient-derived human-induced pluripotent stem cells could provide a better in vitro model to understand AKAP9 functions at different stages of disease progression. In addition, the mechanistic details of how the I2558 M mutation promotes hyperphosphorylated tau by disrupting PP2A expression and alters the tau interactome remain unclear.
In summary, our study showed that the AD-associated AKAP9 I2558 M mutation results in a significant increase in phosphorylated tau protein at residues S396 and S404 site in a differentiated SH-SY5Y P301L cell line. The presence of this mutation altered the composition of tau-interacting proteins, by increasing interaction of proteins associated with translation, RNA localization and oxidative activity. Importantly, functional experiments confirmed the impaired protein synthesis and excessive oxidative stress in a taudependent manner in AKAP9 mutant cells. Our study provides new insights into the mechanisms of how AKAP9 variants contribute to AD pathogenesis.

ACK N OWLED G M ENTS
We would like to thank the staff members of the Laboratory of Molecular NeuroTherapeutics at Boston University for technical assistances, and GenScript Biotech Corporation (Piscataway, NJ) for CRISPR/Cas9-mediated gene editing service. We also thank BioRender.com to provide resources and templates for the illustrations.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.