Defective phagocytic function of induced microglia-like cells is correlated with rapid progression of sporadic ALS

Background: Microglia play a key role in determining the progression of amyotrophic lateral sclerosis (ALS), yet their precise role in ALS has not been identied in humans. The objective of this study was to identify the functional characteristics of microglia and related factors in patients with sporadic ALS that is rapidly progressing. Methods: After conrming that microglia-like cells (iMGs) induced by human monocytes could recapitulate the main signatures of brain microglia, serial comparative studies were conducted to delineate functional differences in iMGs from patients with slowly progressive ALS [ALS(S), n = 14] versus rapidly progressive ALS [ALS(R), n = 15]. Results: Despite an absence of signicant differences in the expression of microglial homeostatic genes, ALS(R)-iMGs preferentially showed defective phagocytosis and an exaggerated pro-inammatory response to LPS stimuli compared to ALS(S)-iMGs. Transcriptome analysis revealed that the perturbed phagocytosis seen in ALS(R)-iMGs was closely associated with decreased NCKAP1 (NCK-associated protein 1)-mediated abnormal actin polymerization. NCKAP1 overexpression was sucient to rescue impaired phagocytosis in ALS(R)-iMGs. To leverage our ndings and identify biological markers of rapidly progressing ALS, we measured plasma miRNA-214-3p (negative regulator targeting NCKAP1) levels. Post-hoc analysis indicated that decreased NCKAP1 expression in iMGs and a concomitant increase in plasma miRNA-214-3p levels was correlated with rapid progression of ALS. Interpretation: Our data suggest that microglial NCKAP1 may be an alternative therapeutic target in rapidly progressive sporadic ALS. In addition, miRNA-214-3p levels could be a serological biomarker for predicting the speed of disease progression. Transcriptome analysis showing that low NCKAP1 expression is associated with impaired phagocytic function in rapidly progressing ALS-iMGs. Transcriptome analysis of the three groups’ iMGs (HC, ALS(S), and ALS(R)) generated from three HC, four ALS(S), and three ALS(R) patients (group #2). a Venn diagram illustrating the number of and overlap between transcripts that differed signicantly in the three groups according to RNA-seq (Fold Change signicant signicantly altered compared specied analysis on (GO:0006909) comparing differential expression in ALS(S)-iMGs and ALS(R)-iMGs compared with (Fold and to GAPDH (n = 3). c Relative expression of plasma miR-214-3p and miR-34c-3p in HCs, slowly progressing, and rapidly progressing ALS patients. Each data point represents one participant (HC: n = 3; ALS(S): n = 14; ALS(R): n = 15, all samples from groups #1, #2, #3). d Correlations of the relative expression of miR-214-3p in plasma, miR-34c-3p in plasma, and NCKAP1 in ALS-iMGs with rates of disease progression (delta-FS, points/month). Each data point represents one patient (miR-214-3p and miR-34c-3p in plasma: n = 29; NCKAP1 in ALS-iMGs: n = 13). p-values were obtained by Pearson correlation. e Correlations of the relative expression of miR-214-3p and miR-34c-3p in plasma with the relative expression of NCKAP1 in ALS-iMGs. Each data point represents one patient (n = 13). f Schematically summarized graphic pathway showing the possible microglial mechanism of rapidly progressing ALS. Briey, actin polymerization could be altered by reduction of NCKAP1 expression, which might be associated with increased levels of miR-214-3p and miR-34c-3p within microglia, resulting in defective microglial phagocytosis. In (b-c), data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, NS: not signicant (one-way ANOVA).

model was shown to exhibit the signature gene patterns of brain microglia and the innate functions of microglia in healthy donors. To further validate the model, we compared iMGs to brain microglia that were obtained from the same ALS patient. Thereafter, we conducted a systematic comparative analysis to delineate the different natures of iMGs in ALS patients according to speed of clinical progression. We enrolled two distinct sporadic ALS patient groups dichotomized by clinical progression speed: one group with slowly progressing ALS [ALS(S), n = 14] and one group with rapidly progressing ALS [ALS(R), n = 15] according to the revised El Escorial criteria between September 2015 and July 2017. Second, we endeavored to identify target molecule(s) related to the functional properties of microglia that are present only in ALS(R)-iMGs. To do this, we compared transcriptome data between ALS(R)-iMGs and ALS(S)-iMGs. Subsequently, we conducted functional studies on an identi ed target molecule. Finally, we found a clinically applicable serologic biomarker related to activity of the target molecule that is well correlated with both levels of the target molecule and speed of progression of ALS.

Participants and Samples
We enrolled ve healthy volunteers and twenty-nine patients with sporadic ALS (14 patients with slow progression and 15 patients with rapid progression) according to the revised El Escorial criteria [29] between September 2015 and July 2017. Patients with clinically de nite, clinically probable, clinically probable with laboratory-supported, or possible sALS were recruited for this study. None of the participants had any evidence of recent infectious or in ammatory diseases. Individual medical records were reviewed to obtain clinical characteristics such as age, sex, family history of ALS, region of symptom onset, ALS functional rating scale-revised (ALSFRS-R) score [29], and changing pattern of ALSFRS-R score re ecting the speed of progression of ALS. The progression rate was de ned as delta FS [30], (i.e., (48 -ALSFRS-R score at the time of diagnosis)/(duration from onset to diagnosis in months)). In order to enroll only participants who exhibited extremely slow or extremely rapid clinical progression, the sALS patients were categorized by using Hanyang MND registry as follows: The mean value was 0.83±0.81, slowly progressive ALS = delta FS ≤ 0.36, and rapidly progressive ALS = delta FS > 1.0. The difference between delta FS value (from onset to diagnosis vs. from onset to at the time of blood sampling) was not signi cant in both groups. The clinical and genetic characteristics of the participants are presented in Additional le 1: Table S1. Schematic outlines of the serial studies that are described in the Methods and Results sections are summarized in Additional le 2: Figure S1. Blood samples were obtained for generation of iMGs and post-hoc analysis of biomarkers, including in ammatory cytokines and microRNAs, after obtaining informed consent from each patient with sALS and matched controls comparable in age and sex at the ALS clinic, Hanyang University Hospital, Seoul, Republic of Korea. This study was conducted in accordance with the World Medical Association's Declaration of Helsinki. It was approved by the Ethics Committee of Hanyang University (HYUH IRB 2013-06-012 and 2017-01-043).

Genetic analysis
Genomic DNA was extracted from peripheral blood leukocytes using a standard procedure. Nextgeneration sequencing (NGS) was performed with SureSelect Human All Exon V5 (SureSelect; Agilent Technologies, Santa Clara, CA) on a NextSeq500 platform (Illumina, Inc.). Alignment of sequence reads, indexing of a reference genome (GRCh37/hg19), and variant calling were performed with a pipeline based on GATK Best Practices. Variants with allele frequencies > 0.01 identi ed in the Genome Aggregation Database (http://gnomad.broadinstitute.org/) were ltered out. Variants found in 1,100 ethnically-matched controls from the Korean Reference Genome Database (http://152.99.75.168/KRGDB/) were also ltered out. Next, 46 genes related to frontotemporal dementia (FTD), ALS, and other dementias were screened for pathogenic or likely pathogenic variants (Additional le 1: Table S1 and Additional le 3: Table S2). These variants were classi ed according to the guidelines of the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP). APOE genotype was also analyzed using whole-exon sequencing data (Additional le 1: Table S1).

Establishment of induced microglia-like cells (iMGs) from human peripheral blood
iMG cells were established based on a method previously published by Ohganani [27]. Brie y, peripheral blood was collected from healthy adult volunteers and ALS patients using a heparinized tube. PBMCs were isolated by density gradient centrifugation using Ficoll (GE Healthcare), according to our previous study [31]. The cells were resuspended in RPMI-1640 (Gibco, Waltham, MA) containing 10% FBS (Gibco) and 1% antibiotic/antimycotic (Invitrogen, Carlsbad, CA). PBMCs were plated in culture chambers at a density of 500,000 cells/ml and cultured overnight under standard culture conditions (37°C, 5% CO 2 ). On the next day, the medium was carefully aspirated and adherent cells (monocytes) were cultured in RPMI-1640 Glutamax (Gibco) supplemented with 1% antibiotic/antimycotic, recombinant human granulocytemacrophage colony-stimulating factor (GM-CSF) ( patterns in iMG cells after treatment with LPS, IL-4, dexamethasone, or during  phagocytosis, we analyzed HLA-DR (PPH00857F), CD45 (PPH01510C), TNF-α (PPH00341F),  Step One Plus TM system (Life Technologies) at 95°C for 10 min, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. A melting curve was generated to examine the speci city of ampli cation. Relative quantity (RQ) levels were calculated with the 2 −ΔΔCt method using GAPDH as an internal standard control.
The reported results are based on three independent experiments carried out on separate batches of cells.

Phagocytosis assay
To quantify phagocytosis in iMGs, iMGs that had been optimally cultured for 21 days were treated with 4 µl red uorescent latex beads for 24 h at 37°C. Phagocytic activity was halted with the addition of 2 ml ice-cold PBS. The cells were washed twice with ice-cold PBS, xed, stained with a microglial marker (IBA-1 or P2RY12), and counterstained with DAPI. Cells were analyzed by confocal microscopy (TCS SP5, Leica). The number of phagocytized beads per IBA-1-positive or P2RY12-positive cell was counted using image J software for phagocytic activity [34]. To assess phagocytosis cup formation in iMGs, cells were xed by adding latex beads for 2 h. For live cell imaging of phagocytosis, iMG cells were grown in imaging dishes (Chamber Slide Lab-Tek II 4; Fisher) and labeled with 100 nM SiR-actin dye (for cytoskeleton staining; Cytoskeleton Inc., North America, USA) according to the manufacturer's protocol [35]. After washing twice with PBS, the old medium was replaced with fresh medium. Three microliters of latex beads (1.1 μm, Sigma-Aldrich) were added to the cells before analysis. Images were captured at a rate of one frame every 1 min 30 sec over a 5 h period. Live imaging was performed using a DeltaVision uorescence microscopy system (Applied Precision) installed at the Hanyang Center for Research Facilities.
Isolation of human brain microglia from the neural tissue of sALS patients We con rmed the microglia signature of iMGs that originated from monocytes from an sALS patient whose blood sample was collected just one day before death. We immediately isolated microglia from fresh brain tissue (brain-MG) from the same patient. The patient had no known pathogenic mutations, including FUS, C9orf72, SOD1, ALS2, SPG11, UBQLN2, DAO, GRN, SQSTM1, SETX, MAPT, TARDBP, or TAF15 gene mutations. For brain-MG culture, the immediately-obtained fresh middle temporal gyrus was washed in HBSS. The tissue was then diced into ~1 mm 3 pieces using a sterile scalpel and transferred to a 50 ml falcon tube containing 10 ml enzyme dissociation mixture with 10 U/ml DNase (Invitrogen) and 2.5 U/ml papain (Worthington, NJ, USA) in Hibernate-A medium (Gibco) (per gram of tissue). The mixture was incubated at 37°C for 10 min with gentle rotation. The tissue was removed from the incubator, gently triturated to aid digestion, and returned to the incubator for a further 10 min. Dissociation was slowed by adding equal volumes of Dulbecco's modi ed Eagle medium and F-12 medium (DMEM/F12; Gibco) with 1% B27 (Gibco). The cell suspension was passed through a 70 μm cell strainer (Bector Dickinson, NJ, USA). Cells were centrifuged at 160 × g for 10 min. The supernatant was discarded and the cell pellet was resuspended in 20 ml DMEM/F12 with 1% B27, 1% GlutaMAX (Gibco), and 1% penicillin-streptomycinglutamine (PSG; Gibco). Next, one-third volume of cold Ficoll (GE Healthcare, Little Chalfont, UK) was added to the cell suspension, and the tube was centrifuged at 4000 rpm for 30 min at 4°C. The interphase containing the microglia was transferred to a new tube (the myelin and erythrocyte layers were discarded) and washed twice with DMEM supplemented with 10% FCS, 1% Pen/Strep, 1% gentamycin, and 25 mM HEPES (Invitrogen). Negative selection of granulocytes (previous method only) and positive selection of microglia with anti-CD15-and anti-CD11b-conjugated magnetic microbeads (Miltenyi Biotec), respectively, were performed by magnetic activated cell sorting (MACS) according to the manufacturer's protocol [36]. Brie y, cells were incubated with 10 μl CD15 microbeads for 15 min at 4°C, washed, suspended in bead buffer (0.5% BSA, 2 mM EDTA in PBS, pH 7.2), and transferred to an MS column placed in a magnetic holder. The ow-through containing unlabeled cells was collected, washed, and incubated with 20 μl CD11b microbeads for 15 min at 4°C. The cells were then washed and placed on a new MS column in a magnetic holder. The CD11b + cell fraction was eluted by removing the column from the magnet, adding bead buffer, and emptying the column with a plunger. Acutely isolated primary microglia were suspended in Trizol reagent (Invitrogen) and stored at -80°C.
We isolated monocytes from the blood of the same patient using anti-CD14-conjugated magnetic microbeads (Miltenyi Biotec) according to the manufacturer's protocol. The isolated monocytes were suspended in Trizol reagent (Invitrogen) and stored at -80°C for RNA-seq and qPCR.

RNA sequencing and data analysis
Total RNA was isolated using Trizol reagent (Invitrogen). RNA quality was assessed with an Agilent 2100 bioanalyzer using an RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, Netherlands). Control and test RNA libraries were constructed using a SENSE 3′ mRNA-Seq Library Prep Kit (Lexogen, Inc., Austria) according to the manufacturer's instructions. The library was ampli ed to add complete adapter sequences required for cluster generation. The constructed library was puri ed from PCR components. High-throughput sequencing was performed as single-end 75 sequencing using a NextSeq 500 platform (Illumina, Inc., USA). SENSE 3′ mRNA-Seq reads were aligned using Bowtie2 version 2.1.0 [37]. Bowtie2 indices were generated either from genome assembly sequences or from representative transcript sequences aligned with the genome and transcriptome. The alignment le was used to perform transcript assembly, estimate gene abundance, and detect differential gene expression. Differentially expressed genes (DEGs) were determined based on counts from unique and multiple alignments using EdgeR in R version 3.2.2 and BIOCONDUCTOR version 3.0 [38]. Read count (RT) data were processed based on the global normalization method using Genowiz™ version 4.0.5.6 (Ocimum Biosolutions, India). Gene classi cation was based on searches performed in the DAVID (http://david.abcc.ncifcrf.gov/) and Medline (http://www.ncbi.nlm.nih.gov/) databases. We used MeV 4.9.0 to perform sample and gene clustering and to visualize gene clusters and heat maps. Hierarchical cluster analyses were performed using Euclidean distance as a similarity measurement with average linkage heuristic.
Cell culture

Enzyme-linked immunosorbent assay
Secretion of pro-and anti-in ammatory cytokines (TNF-α, IL-1β, IL-6, IL-10, and TGF-β1) during LPS stimulation from culture supernatants was tested using a commercially available cytokine assay kit obtained from Millipore (

Plasma miRNA analysis
The small RNA-enriched fraction was extracted from 625 μl of the plasma sample using a mirVana miRNA isolation kit, following the manufacturer's instructions (Ambion, Austin, TX). The purity of the extracted RNA was quanti ed using a NanoDrop™ 1000 spectrophotometer. For reverse transcription and quantitative real-time PCR, a xed volume of 5 μl of the small RNA-enriched fraction obtained from a given sample was used for the reverse transcription (RT) reaction. For synthesis of each miRNA-speci c cDNA, miRNA was reverse transcribed using the TaqMan miRNA reverse transcription kit (Life Technologies min. Data analysis was performed to determine the threshold cycle (Ct). Relative quantities of miRNA were calculated using the 2 −ΔΔCt method after normalization to hsa-miR-16 levels in plasma samples.

Statistical analysis
Data are presented as mean ± SEM. The statistical signi cance of differences between groups was assessed with Student's t-test, one way-ANOVA, and two way-ANOVA using GraphPad Prism 7 (GraphPad Software, San Diego, CA). The ndings were regarded as signi cant when *p < 0.05, **p < 0.01, ***p < 0.001, and **** p < 0.0001. In post-hoc analysis, p-values were obtained by Pearson correlation.

Healthy control (HC)-iMGs present the main signatures of microglia and show intrinsic functions
As shown in the schematic diagram of our successive experiments (Additional le 2: Figure S1), we generated human iMGs from PBMCs obtained from ve healthy controls (HCs) treated with inducible supplements consisting of IL-34 and GM-CSF; the PBMCs were cultured for 3 weeks to induce a more mature form of microglia-like cells. The generated iMGs displayed well-rami ed morphology after 21 days in culture (Fig. 1a, Additional le 4: Figure S2). They were conventionally identi ed as CD11b + CD45 low cells by ow cytometry analysis (Fig. 1b). They were characterized by upregulated expression of resident microglia surface markers including P2RY12 and IBA-1 in immuno uorescence analysis. The monocyte marker CCR2 disappeared from iMGs (Fig. 1c). In qRT-PCR ndings, key signatures of microglia, such as P2RY12, OLFML3, TGFBR1, and TREM2, were signi cantly upregulated in iMGs compared to monocytes (Fig. 1d). mRNA levels of in ammation-related factors were altered in iMGs upon induction with IL-4, LPS, or dexamethasone, which is consistent with observations from a previous postmortem microglial study (Fig. 1e) [32]. In addition, iMGs showed normal phagocytic function and increased expression of TNF-α mRNA upon stimulation with latex beads ( Fig. 1f and g). The optimal culturing time period and minimal blood sample amounts to be used in serial subsequent studies for the preparation of iMGs from ALS patients were identi ed based on trial and error data achieved from the HC-iMGs model.
Although our data and the results of previous studies [23,26] suggest that HC-iMGs present key signatures of microglia and show intrinsic microglial functions, it is unclear whether iMGs derived from PBMCs accurately re ect brain microglia in the same person. To address this key question, blood was obtained from a patient with sALS just before death who consented to his body being donated. Brain microglia from that patient were immediately isolated from autopsy tissue using CD11b beads. These samples were used in the next experiment.
iMGs express key genetic signature of brain microglia To identify similarities between iMGs and brain microglia, we obtained fresh brain tissue and peripheral blood from a patient with sALS, and then performed RNA sequencing (RNA-seq) of iMGs, brain microglia (brain-MG, CD11b + ), and monocytes (CD14 + ) (Fig. 2a). Whole-transcriptome differential gene expression analysis revealed 13,038 genes that had a greater than 3-fold difference in either iMGs or brain-MG compared to monocytes. Of these, 705 (5.4%) overlapped between iMGs and brain-MG (Fig. 2b, Additional le 5: Table S3). In comparison, only 195 (1.5%) of the enriched genes overlapped between iMGs and monocytes. Hierarchical clustering analysis revealed similarities between iMGs and brain-MG ( Fig. 2c). Through gene ontology (GO) analysis of the 705 overlapping genes, we found ten signi cant pathway modules that were commonly expressed in both iMGs and brain-MG. These represent gene subsets related to extracellular matrix organization, metabolic processes, oxidation-reduction processes, cell migration, and in ammatory responses (Fig. 2d, Additional le 5: Table S4). Searching the Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed ECM-receptor interactions, metabolic pathways, and focal adhesion (Fig. 2d, Additional le 5: Table S4). Regarding microglial signature genes [39], the expression levels of SPP1, JUN, TREM2, APOE, HEXB, MEF2A, LILRB4, CX3CR1, ITGAX, TGFBR1, P2RY12, MAFB, TGFB1, and SLCO2B1 in iMGs were similar to those in brain-MG, although the expression levels of OLFML3, AXL, CSF1R, RHOB, EGR1, and TMEM119 in iMGs were relatively low (Fig. 2e).
Immuno uorescence staining showed that P2RY12, IBA-1, and the transcription factor PU.1 were well preserved in both iMGs and brain-MG, although immunoreactivity (IR) of TMEM119 in iMGs was less than in brain-MG (Fig. 2f).

ALS(R)-iMGs show dystrophic morphology and severely impaired phagocytic function
To verify our hypothesis that microglial dysfunction was associated with the speed of progression of ALS, we generated iMGs from three HCs (C1 -C3) and eleven patients with sALS [6 slowly progressing ALS(S) (S1 -S6) and 5 rapidly progressing ALS(R) (R1 -R5) patients]. Their demographic characteristics are summarized in Additional le 1: Table S1. The participants enrolled in this rst step study were assigned into "group #1" (Additional le 2: Figure S1). To exclude the possibility of genetic effects on ALS progression, sALS patients were only enrolled after con rming that they had no familial history of ALS or ALS-related major known genetic mutations based on whole exome sequencing (WES). The genetic panel included SOD1, ALS2, TDP-43, FUS, C9orf72, and OPTN (Additional le 3: Table S2).
The expression of genes related to homeostatic microglial function (P2RY12, OLFML3, TGFBR1, GPR34, MERTK, HEXB, and CSF1R, and TMEM119) was not signi cantly different between ALS(S)-iMGs and ALS(R)-iMGs, although the expression levels of several genes were decreased compared to those in HC-iMGs (Fig. 3a). Immunocytochemical staining showed that P2RY12, a representative microglia-speci c gene signature [40], was well preserved in both iMGs from both ALS groups (Fig. 3b). Despite the lack of expression level differences in microglial signature genes between iMGs from the two ALS groups, Imarisbased morphometric analysis revealed that ALS(R)-iMGs were signi cantly different from ALS(S)-iMGs and HC-iMGs (Fig. 3c). Based on morphological parameters for dynamic microenvironment surveillance, dendritic length, number of segmentations, branching, and dendritic terminal points were signi cantly reduced in both ALS group-iMGs compared to HC-iMGs. However, the number of branches and terminal points were markedly reduced in ALS(R)-iMGs compared to ALS(S)-iMGs, although there was no difference in cell area of iMGs between the two ALS groups and HC. Furthermore, phagocytic function was compared between ALS(S)-iMGs and ALS(R)-iMGs. The most remarkable nding was that phagocytic function was severely impaired in ALS(R)-iMGs, whereas ALS(S)-iMGs exhibited no signi cant differences in phagocytic function compared to HC-iMGs (Fig. 3d, e, and Additional le 6: Figure S3).
Next, to exclude the possibility of senescence-related factors leading to phagocytic dysfunction of microglia [41], we compared the expression levels of senescence-related genes using qRT-PCR. There was no difference between ALS(S)-iMGs and ALS(R)-iMGs in the expression of the cellular senescence markers p21 CIP1 and p16 INK4 , and there was no difference in age at sampling time between the patient groups (Fig. 3f, Additional le 2: Table S1). Thus, we ruled out aging as a factor in the defective phagocytic function shown in ALS(R)-iMGs Defective phagocytosis in ALS(R)-iMGs is associated with decreased NCKAP1 expression Next, we performed comparative transcriptome analysis to identify molecular targets involved in phagocytic dysfunction of ALS(R)-iMGs after generating a second set of iMGs (group #2, Additional le 2: Figure S1) from four patients with ALS(S) (S7 -S10), three patients with ALS(R) (R6 -R8), and three HCs (C1 -C3). The second set of ALS(R)-iMGs also showed phagocytic dysfunction (Additional le 6: Figure S3).
Whole-transcriptome differential gene expression analysis revealed 4,367 genes with a 1.5-fold difference in expression in this analysis (Fig. 4a). The proportion that overlapped between ALS(R)-iMGs and ALS(S)-iMGs was 19.7% (861 genes). GO analysis of those 861 genes revealed that they were related to immune response, regulation of NF-κB transcription factor activity, innate immune response, etc. (Additional le 5: Table S5). Importantly, we found that 2,559 transcripts were signi cantly different between ALS(R)-iMGs and ALS(S)-iMGs (Additional le 5: Table S6). GO analysis of the 2,559 genes revealed gene subsets including those involved in chemotaxis, cilium assembly, long-chain fatty-acyl-CoA biosynthesis, response to lipopolysaccharide, in ammatory responses, actin lament polymerization, immune responses, metabolic processes, the Fc-gamma receptor signaling pathway involved in phagocytosis, and phagocytosis. KEGG pathways included metabolic pathways, focal adhesion, ECM-receptor interactions, ubiquitin-mediated proteolysis, and cytokine-cytokine receptor interactions (Fig. 4b, Additional le 5: Table  S7).
Thus, we speculated that subsets of genes involved in chemotaxis, actin lament polymerization, phagocytosis, focal adhesion, ECM receptor interactions, and immune-related pathways might be associated with defective phagocytosis in ALS(R)-iMGs. When we used GO to focus on phagocytosisrelated pathways and genes, heatmap analysis revealed that genes including NCKAP1, VAV3, MYO10, FYN, ARPCA1, and SLC11A1 were signi cantly down-regulated in ALS(R)-iMGs compared to ALS(S)-iMGs (Fig. 4c). Moreover, MFGE8, PTX3, GAS6, FCGR2B, and CD36 were upregulated in ALS(R)-iMGs. To con rm these results, we analyzed mRNA expression levels of these major genes using all available samples from both group #1 and #2 (Fig. 4d). Interestingly, NCK-associated protein 1 (NCKAP1) and Guanine Nucleotide Exchange Factor Vav 3 (VAV3), known intracellular signaling-regulated factors related to the actin-polymerization process after the initial recognition step in phagocytosis [42], were signi cantly decreased in ALS(R)-iMGs. Moreover, ALS(R)-iMGs had decreased TREM2 levels, which may partially contribute to defects in phagocytic function. However, levels of MFGE8 and PTX3, recognition and enhancing factors in phagocytosis, were elevated.
As shown in Fig. 4c-e, the transcript showing the greatest difference in ALS(R)-iMGs was NCKAP1, which is a key component of the actin polymerization process related to the phagocytic machinery. The low level of NCKAP1 mRNA noted in ALS(R)-iMGs does not re ect an intrinsic characteristic of monocytes, which rarely express NCKAP1 [43]. This nding was supported by the data showing high expression of NCKAP1 mRNA in ALS(S)-iMGs (Fig. 4e). Additionally, NCKAP1 was highly expressed in HMC3 cells and iMGs compared to monocytes (Additional le 7: Figure S4). This result suggests that NCKAP1 is the most important factor related to defective phagocytosis. Thus, we further investigated the critical role of the NCKAP1 gene in phagocytosis. In the next step, we generated another new set ("group #3") of iMGs from four patients with ALS(S) (S11 -S14) and seven patients with ALS(R) (R9 -R15) (Additional le 2: Figure S1).

NCKAP1 regulates actin polymerization during the phagocytic process in iMGs
To further delineate the role of NCKAP1 in phagocytosis, ALS(R)-iMGs (R9 -R15) and ALS(S)-iMGs (S11 -S14) were transfected with GFP vector, shNCKAP1-GFP, or NCKAP1-GFP and subsequently cultured for 72 hours. The e ciency of NCKAP1 knockdown in HeLa cells and iMGs was evaluated by qRT-PCR (Additional le 8: Figure S5a and b). Knockdown was also con rmed by immuno uorescence analysis (Additional le 8: Figure S5c). NCKAP1-GFP-transfected cells showed intense IR of phalloidin, a lamentous actin (F-actin) marker, which was absent from shNCKAP1-transfected cells. These ndings suggest that NCKAP1 is involved in the formation of F-actin. Similar ndings were reproduced in both ALS(S)-and ALS(R)-iMGs (Additional le 8: Figure S5d). Decreased levels of NCKAP1 and phalloidin IR were noted in ALS(R)-iMGs compared to ALS(S)-iMGs (upper gures). However, transduction of shNCKAP1 reduced IR of both phalloidin and NCKAP1 in ALS(S)-iMGs, and the opposite was true in NCKAP1-GFP-transfected ALS(R)-iMGs (lower gures).
NCKAP1 is a known member of the WAVE regulatory complex (WRC) [44]. Thus, we investigated the relationship between NCKAP1 and other actin polymerization-related genes and the effect of NCKAP1 overexpression or knockdown on the expression of related molecules such as cytoplasmic FMR1interacting protein 1 (CYFIP1), Abelson interactor 2 (ABI2), WASP-family verprolin homologous protein 1 (WAVE1), and WAVE2 using iMGs from "group #3." As shown in Fig. 5a Fig. 5a). Immuno uorescence revealed that WAVE complexes, such as WAVE and ABI, co-localized with NCKAP1 in ALS(S)-iMGs but not in ALS(R)-iMGs ( Fig. 5a and b). Next, to evaluate the role of NCKAP1 in WAVE complex stability, we transfected HeLa cells with GFP-tagged NCKAP1 or GFP-tagged NCKAP1 shRNA and then performed Western blots to determine the effects of NCKAP1 on actin polymerization-related proteins. We found that NCKAP1 overexpression increased expression of actin polymerization-related proteins (CYFIP1, ABI2, WAVE1, and WAVE2), whereas NCKAP1 knockdown reduced their expression ( Fig. 5c and d).
Finally, we examined whether NCKAP1 overexpression rescued the defective phagocytic function of ALS(R)-iMGs using live cell imaging. While phagocytosis of latex beads was clearly present in ALS(S)-iMGs (upper part of Fig. 5e), ALS(R)-iMGs showed defective phagocytosis (bottom part of Fig. 5e). On the contrary, when NCKAP1 was overexpressed using NCKAP1-GFP in ALS(R)-iMGs, phagocytosis was rescued. As expected, the active phagocytosis seen in ALS(S)-iMGs was not present when NCKAP1 was knocked down with shNCKAP1-GFP. These ndings are evident in snapshot images ( Fig. 5e and f) and live cell images (Additional le 9-12: Video S1-4). Collectively, our data suggest that NCKAP1 plays a pivotal role in the formation of phagocytic cups by participating in the WAVE complex-mediated actin polymerization process. Thus, NCKAP1 is an important potential biomarker that could be useful for predicting the state of perturbed phagocytic function of iMGs in rapidly progressing ALS patients.

ALS(R)-iMGs have an exaggerated response to in ammatory signaling
Phagocytosis is traditionally regarded as bene cial for tissue homeostasis; it is responsible for rapid clearance of dying cells or debris, thus preventing spillover of pro-in ammatory and neurotoxic responses [42]. Transcriptome analysis showed that the immune response pathway, which operates in response to LPS and in ammatory signaling, functions differently in iMGs from the two ALS groups, as shown in Fig.   4b. Thus, we compared the mRNA expression levels of cytokines in response to LPS stimulation in ALS(S)-iMGs and ALS(R)-iMGs in "group #1" samples after con rming that our culture environment could mirror the differential phenotypes [Additional le 13: Figure S6]. In the unstimulated state, there was a signi cant difference in mRNA levels of the cytokines (TNF-α, IL-6, TGF-β1, and IL-10) between both ALS groups and monocytes, and the difference was reproduced in iMGs [Additional le 14: Figure S7]. In addition, LPS stimulation provoked an increase in mRNA expression of pro-in ammatory cytokines and a decrease in TGF-β expression in iMGs from both ALS groups. ALS(R)-iMGs exhibited an exaggerated TNF-α, IL-6, IL-1β, and TGF-β response compared to ALS(S)-iMGs upon LPS stimulation (Fig. 6a). Cytokine levels in the culture media showed a pattern that was similar to the mRNA expression pro les, as measured by ELISA (Fig. 6b). These ndings suggest that the response of ALS(R)-iMGs to an in ammatory stimulus is exaggerated in comparison to the response of ALS(S)-iMGs.
To address whether the enhanced pro-in ammatory response seen in ALS(R)-iMGs is associated with decreased NCKAP1 expression, we examined the causal relationship between key in ammatory signals and NCKAP1. Because we had no frame of reference regarding the role of NCKAP1 in microglial function and in ammatory signaling, we speculated that NCKAP1 might show similar acting with NCKAP1L (NCKAP1-like). NCKAP1L and NCKAP1 belong to the same family and have similar structures. NCKAP1L is known as a crucial player in actin polymerization. It is selectively expressed in hematopoietic cells [45].
Recently, NCKAP1L was proposed to be a novel phagocytosis regulator in a phagocyte cell line [46].
Furthermore, NCKAP1L is a common upstream signal with NF-κB, which is a representative in ammatory signal in hematopoietic cells [47]. Thus, we studied whether NCKAP1 reduction is involved in NF-κB signaling in ALS(R)-iMGs. We examined NF-κB signaling in response to LPS stimulation in ALS(R)-iMGs. NF-κB p-50 and p-65 mRNA expression levels were strongly upregulated in ALS(R)-iMGs upon LPS stimulation (Fig. 6c). Overall, our results indicate that NCKAP1 reduction may be related to the abnormally exaggerated in ammatory response via the NF-κB signaling pathway. This nding provides a clue as to why an enhanced pro-in ammatory response is present in ALS(R)-iMGs.
In sum, we concluded that NCKAP1 reduction induced defective phagocytic function and exaggerated the pro-in ammatory response in ALS(R)-iMGs. In addition, NCKAP1 levels in iMGs could re ect the state of clinical progression of ALS. However, measurement of NCKAP1 activity is possible only after a timeconsuming process of generating iMGs. Thus, it cannot be used as a marker for the speed of progression of ALS in a practical context. To nd more useful possible biomarkers related to NCKAP1 activity that can predict the speed of clinical progression of ALS, serial subsequent studies were undertaken. After rechecking that no NCKAP1 mutations or pathogenic polymorphisms were detected in the WES results of 29 ALS patients (Additional le 1: Table S1), we searched for serologically available biomarkers related to NCKAP1 expression.
miRNA-214-3p and miRNA-34-3p targeting NCKAP1 are associated with NCKAP1 reduction After excluding the possibility of NCKAP1 mutations, we searched for miRNA candidates that regulate NCKAP1 expression. miRNA targets were predicted by combinatorial utilization of two different web-based prediction algorithms, TargetScan and miRanda. We also collected targets that had been experimentally con rmed in vitro along with ALS-related and in ammation-related miRNAs from published studies. miR-214-3p and -34c-3p were selected as candidates. The two micro-RNAs can directly reduce NCKAP1 expression [48,49]. Herein, we identi ed a putative miR-214-3p and -34c-3p binding site in the 3′-UTR of NCKAP1 using bioinformatics tools for miRNA target prediction (Fig. 7a). To determine whether miR-214-3p and -34c-3p directly target NCKAP1, the effects of miR-214-3p and -34c-3p mimics and inhibitors were tested in HeLa cells for their effects on NCKAP1 gene expression. As shown in Fig. 7b, both miR-214-3p and -34c-3p downregulated NCKAP1, while the inhibitors upregulated NCKAP1. Both miRNAs were strongly expressed in iMGs (Additional le 7: Figure S4). This nding indicates that both miR-214-3p and miR-34c-3p play a role in regulating NCKAP1 levels.
Based on the serial experimental results, we selected NCKAP1 and its regulators (miR-214-3p and -34c-3p) as candidate biomarkers that re ect the phagocytic function of ALS-iMGs and the clinical progression of ALS. To validate our ndings, post-hoc comparative analysis was done to address whether clinical data on the enrolled participants (groups #1, 2, 3) correlated with the selected miR candidates. As shown in Fig. 7c, plasma levels of miR-214-3p and -34c-3p were increased in both ALS groups compared to HCs. Additionally, both miRNAs were signi cantly increased in ALS(R) compared to ALS(S) (plasma miR-214-3p: p = 0.043; plasma miR-34c-3p: p = 0.021).
Finally, we analyzed the relationship between each patient's speed of clinical progression, expressed as delta-FS (points/month), and the activity of the three biomarkers (NCKAP1, miR-214-3p, and miR-34c-3p).
In summary, our data indicate that the perturbed phagocytic function seen in ALS(R)-iMGs is related to decreased NCKAP1-mediated impairment of proper actin polymerization. Regarding serological biomarkers related to NCKAP1, post-hoc analysis indicated that delta-FS, representing the speed of progression of ALS, correlates well with each patient's biomarkers, including NCKAP1, plasma miRNAs, and in ammatory cytokines. Thus, targeting microglial NCKAP1 may be an alternative therapeutic target in rapid sALS. In addition, miRNA-214-3p could be a serologic biomarker that is clinically useful for predicting the speed of progression of ALS. Our current hypothesis regarding the involvement of the microglial pathway in rapid progression of ALS is summarized graphically in Fig. 7f.

Discussion
The signi cance of the current study is that we identi ed serologically reliable biological markers and possible therapeutic targets associated with defective microglial functioning in rapidly progressing ALS using an induced microglia-like cell model. Using transcriptome analysis, we identi ed defective phagocytosis corresponding with reduced NCKAP1 levels as the key factor that gave rise to phagocytic dysfunction in ALS(R)-iMGs. Though there was no signi cant difference in major homeostatic gene pro les between the two groups of ALS-iMGs, as shown in Fig. 3a, NCKAP1 levels were reduced in both groups of ALS-iMGs as compared with HC-iMGs. Only ALS(R)-iMGs showed intrinsically perturbed phagocytosis and an exaggerated in ammatory response via increased NF-κB signaling in response to LPS stimulus. These data imply that NCKAP1 reduction in ALS(R)-iMGs may be responsible for both defective phagocytosis and the accelerated in ammatory response in a pro-in ammatory environment, a well-known condition in ALS. This result is supported by data on ALS(S)-iMGs showing intact phagocytic function with less of a pro-in ammatory response than is present in ALS(R)-iMGs.
Given that activated in ammatory responses and excessive oxidative stress are commonly encountered environments in ALS, it is understandable that previously reported data suggests that the progression of ALS is correlated with the activity of pro-in ammatory cytokines [12,50]. These ndings agree with our post-hoc data, which revealed that rapidly progressing ALS patients with perturbed phagocytosis in their iMGs also showed an increase in plasma levels of the pro-in ammatory cytokine IL-8 compared to the slowly progressing ALS group, as shown in Additional le 15: Figure S8. Moreover, the rate of disease progression (delta FS) was also correlated with the level of plasma IL-8 (Additional le 15: Figure S8f).
We looked at the steps of the phagocytic process using transcriptome analysis and found that mRNA levels of MFGE8, a recognition receptor [51], and PTX3, an enhancer of microglial phagocytic activity [52], were signi cantly higher in ALS(R)-iMGs despite their defective phagocytic functioning. Another interesting nding was that expression of TREM2, a well-known risk gene related to microglial phagocytic function in Alzheimer's disease (AD) [53,54], Parkinson's disease (PD) [55], and ALS [56], was decreased in ALS(R)-iMGs, as shown in Fig. 4d. These data also imply that compensatory mechanisms that are activated in response to perturbations in phagocytosis work by elevating the levels of recognition molecules (MFGE8 mRNA) or by enhancing microglial phagocytic activity, such as that involving PTX3 mRNA; concomitant decreases in TREM2 activity and NCKAP1-mediated improper actin polymerization may contribute to the defective phagocytic function seen in ALS(R)-iMGs.
Only a few studies have reported an association between NCKAP1 and neurodegenerative diseases; for example, NCKAP1 gene expression is known to be reduced in AD [57]. Most studies of NCKAP1 have focused on its role in neuronal differentiation and migration as a cytoskeletal regulator during development [58]. Furthermore, NCKAP1L, a hematopoietic cell-speci c gene that has a similar structure to NCKAP1, has been reported as a key player in actin polymerization. It is also essential for neutrophil and macrophage migration and phagocytosis [43,45,46]. NCKAP1L family members are known to regulate actin polymerization, morphogenesis, and immunity [45]. Both NCKAP1 and NCKAP1L are members of the WRC that consists of Abi (Abelson interactor 1 or 2), WAVE (WAVE 1, 2, 3), Sra1 (speci cally Rac-associated protein 1), and activated Arp2/3 (actin-related protein-2/3), all of which can promote actin polymerization. In contrast to NCKAP1L, NCKAP1 is enriched in the brain, but absent or less expressed in hematopoietic cells [47]. Our results showed that ALS(R)-iMGs overexpressing NCKAP1 exhibited restored phagocytic function and increased expression levels of CYFIP1, ABI2, WAVE1, and WAVE2. Because the stability of WRC is interdependent on the presence of individual WRC components [45], low NCKAP1 expression may cause instability of the complex and hinder F-actin polymerization in microglia-like cells. Therefore, NCKAP1 is presumed to play a key role in the engulfment process of phagocytosis by regulating actin cytoskeleton dynamics.
Numerous studies have focused on toxic microglia as a factor in ALS progression. Reactive microglia can aggravate motor neuron death through pro-in ammatory cytokine secretion in SOD1 mice. Depletion of defective microglial cells can resolve neuroin ammation and result in prolonged survival [59].
Additionally, ALS(R)-iMGs showed an intense pro-in ammatory reaction to LPS stimuli, in accordance with the results of previous SOD1 mouse and human studies [10,60,61]. This reaction is distinct from the general characteristics of aged microglia [39,62]. The exaggerated pro-in ammatory response of ALS(R)-iMGs to LPS stimuli (like immune vigilance) [41] may be associated with reduced expression of NCKAP1 and related WRC genes. In the hematopoietic system, cytokine expression via NF-κB signaling and actin polymerization for phagocytosis have a common upstream signaling molecule, Rac small GTPase.
However, these two pathways bifurcate upstream of Rac [45]. Thus, Rac activation by LPS can induce both NF-κB signaling and phagocytosis. However, reduced expression of NCKAP1 and the resulting decrease in actin polymerization-related proteins may shift Rac-GTP signaling toward NF-κB signaling, causing NF-κB over-activation and increased levels of pro-in ammatory cytokines in microglia. Our results support this speculation. ALS(R)-iMGs exhibited lower NCKAP1 expression but higher NF-κB expression upon LPS stimulation than did ALS(S)-iMGs. Similar ndings were observed in neurodegeneration-associated molecular patterns (NAMPs) [63], including mutant SOD1, FUS, TDP-43, RNA foci and RAN dipeptides, and degenerating neuronal debris. NAMPs may trigger a chronic in ammatory milieu in CNS, while microglia acting under acute in ammatory conditions in response to LPS have distinct activation pro les [64].
We searched for biological markers that could predict the speed of progression of ALS, and selected miRNA-214-3p and miRNA-34c-3p as reliable candidates. Both can directly reduce the level of NCKAP1 expression [48,49], in agreement with our results. Speci cally, miRNA-214-3p expression was increased in microglia isolated from SOD1-G93A mice [65] and in muscles [66] and sera [67] of ALS patients. This miRNA acts as an in ammatory mediator that induces NF-κB activation and IL-6 expression [68]. miRNA-34c-3p also represses the expression of sirtuins (SIRTs). SIRT1 inhibits NF-κB activation upon in ammation in neurodegenerative disease states [69]. Our results suggest that the increased expression of miRNA-34c-3p and miRNA-214-3p that is seen in the plasma of patients was associated with rapidly progressing ALS, resulting in possibly targeting microglial NCKAP1. Thus, NCKAP1 and miRNA-214-3p could be alternative therapeutic targets and reliable biological markers that re ect the speed of progression of ALS.
Our study leaves several unanswered questions. Although we propose the utility of the serological biomarkers miRNA-214-3p and miRNA-34c-3p, which inhibit NCKAP1 expression in an in ammatory environment, we did not clarify how NCKAP1 was reduced in only ALS(R)-iMGs in our culture environment. One possible mechanism is through maintenance of epigenetic memory during direct conversion [70]. Direct conversion methods, including ours, have an advantage in that they preserve the aging-associated features of the donors, while iPSC models alter the epigenetic landscape during rejuvenation [71]. Further studies are needed to clarify whether our model maintains epigenetic memory.
The next question is whether our culture media re ect CNS microenvironment of ALS patients. To address this issue, we con rmed that our culture environment could form pro-in ammatory environment and which was associated with GM-CSF (10 ng/mL) and IL-34 (100 ng/mL) for iMG generation. In fact, GM-CSF-treated monocytes express an in ammatory phenotype that includes secretion of pro-in ammatory cytokines, in agreement with previous studies [72,73]. Thus, NCKAP1 reduction by miRNA-214 and miRNA-34 in ALS(R)-iMGs is another possible mechanism for the observed defective phagocytic function, although we did not examine miRNA levels in ALS(R)-iMGs owing to the limited quantity of available samples. Another limitation of this study is related to the isolation of brain microglia from ALS patients using CD11b-positive beads that was done in order to compare the brain microglia with our iMGs model. These cells might be a mixture of in ltrated macrophages and resident microglia. In addition, we cannot exclude the possibility that the in ammatory characteristics of monocytes in rapidly progressing ALS patients [50] might contribute to the characteristics of our iMGs, despite the fact that NCKAP1 expression in the monocytes of ALS patients is rare. There is still an argument that the iMGs model is closer to in ltrated blood-derived macrophages than resident microglia [23]. A third limitation is that iMGs are not absolutely identical to resident brain microglia. They cannot precisely re ect the characteristics of yolk sac-originated resident microglia in the non-diseased brain [74]. To overcome these inevitable hurdles of iMGs, studies focusing on the development of new, detailed markers that can discriminate iMGs from or correlate iMGs with microglia subpopulations [9] and diverse macrophage populations are needed. More precise single cell assay-based analytic approaches to the study of iMGs are needed.
Despite these limitations, our results revealed many similarities of iMGs with microglia, including intrinsic phagocytic functions and major signature gene pro les, that were demonstrated in our in vitro model and autopsied brain sample from an ALS patient. In addition, our iMGs model has an advantage in that it can be used to predict current complex in ammatory conditions associated with resident microglial phagocytic functions which iMGs share with in ltrating macrophages/monocytes. The interaction of in ltrated leukocytes with resident microglia could be considered a current outcome or status of neuroin ammation per se for predicting the pathophysiological process and speed of disease progression in ALS patients [13]. Therefore, our iMGs model may be a useful tool for indirectly understanding some aspects of microglial phagocytic function in ALS patients who exhibit different phenotypes. Moreover, this model can provide new insights into the multifaceted nature of microglia and microglial phagocytosis in ALS.

Conclusions
We speculate that the enhanced in ammatory milieu in rapidly progressing ALS patients increases miRNA-214-3p and miRNA-34c-3p levels, leading to reduced expression of NCKAP1 in microglia. This may interfere with the engulfment step of phagocytosis and induce immune vigilance in rapidly progressing ALS. In addition, an increase in plasma levels of miRNA-214-3p was closely correlated with the clinical speed of progression in ALS patients and with NCKAP1 expression in their iMGs. Therefore, NCKAP1-mediated disruptions in phagocytosis may be a good therapeutic target in ALS. Furthermore, our data indicate that miRNA-214-3p is an easily measurable biomarker that re ects the speed of progression of ALS.

Availability of data and materials
The transcriptome analyses performed in iMGs and brain tissue are available upon request.

Con icts of interest
All authors declare that they have no competing interests.  100 μm. b In ow cytometry, most iMGs showed high CD11b and low CD45 expression on day 21. c iMGs expressed CX3CR1 (green) and the speci c microglial markers P2RY12 (red) and IBA-1 (green), but not CCR2 (green), a marker of monocytes. DAPI (blue) was used to visualize nuclei. The gure is representative of several replicates of independent experiments (n = 6). Scale bar: 25 μm. d Microglial signature gene expression pro les (P2RY12, OLFML3, TGFBR1, TMEM119, and TREM2) of iMGs analyzed by qRT-PCR. Fold changes in mRNA expression compared to monocytes. Each dot represents data from one individual's HC-iMGs and monocytes (n = 5 in group #1) e Cytokine expression pro les for iMGs stimulated with LPS, dexamethasone, or IL-4 analyzed by qRT-PCR. Each dot represents data from an individual's HC-iMGs (n = 3 in group #1). f Phagocytic activity of iMGs (IBA-1: green) incubated with uorescent latex beads (red) for 24 h. DAPI (blue) was used to visualize nuclei. The gure is representative of several independent experiments (n = 5). Scale bar: 25 μm. g After phagocytosis for 72 h, TNF-α mRNA expression in iMGs was signi cantly higher than that in controls (vs. without bead). Each dot represents data from an individual's HC-iMGs and monocytes (n = 5 in group #1). In (d-g), data are presented as mean ± SEM. **p < 0.01, ***p < 0.001, NS: not signi cant (Student's t-test or one-way ANOVA).

Figure 2
Transcriptome comparison between iMGs and brain microglia obtained simultaneously from a single ALS patient. a Schematic representation of the experimental procedure used to compare iMGs with brain microglia (brain-MG) and monocytes in a ALS patient. b Venn diagram showing unique and intersecting genes (13,038) that are differentially expressed (DE) in monocytes, iMGs, and brain-MG according to RNA-seq (Fold Change > |3|). c Heatmap of DE genes from gene transcriptome comparisons between monocytes, iMGs, and brain-MG. A pseudo-count was used to obtain FPKM values (FPKM +1), log2transformed, and each gene was normalized in its respective row (n = 13,038). d GO analysis of ten signi cant pathway modules and ve KEGG pathway modules in the 705 genes shared between iMGs and brain-MG. The number within each bar indicates the number of genes in the database for the speci ed term. e Bar graphs of microglial-speci c or -enriched genes measured in iMGs and brain-MG as [log2 (FPKM +1)] presented as mean ± SEM. f Immunocytochemical staining of IBA-1, P2RY12, TMEM119, and PU.1. DAPI was used to visualize nuclei. The gure is representative of independent experiments performed in replicates (n = 10). Scale bar: 25 μm. Transcriptome analysis showing that low NCKAP1 expression is associated with impaired phagocytic function in rapidly progressing ALS-iMGs. Transcriptome analysis of the three groups' iMGs (HC, ALS(S), and ALS(R)) generated from three HC, four ALS(S), and three ALS(R) patients (group #2). a Venn diagram illustrating the number of and overlap between transcripts that differed signi cantly in the three groups according to RNA-seq (Fold Change > |1.5|). b GO analysis of ten signi cant pathway modules and ve KEGG pathway modules from 2,559 transcripts that were signi cantly altered in ALS(R)-iMGs compared with ALS(S)-iMGs. The number within each bar indicates the number of genes in the database for the speci ed term. c Heatmap analysis focused on phagocytosis (GO:0006909) comparing differential gene