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Complement C4 Is Reduced in iPSC-Derived Astrocytes of Autism Spectrum Disorder Subjects

In recent years, accumulating evidence has shown that the innate immune complement system is involved in several aspects of normal brain development and in neurodevelopmental disorders, including autism spectrum disorder (ASD). Although abnormal expression of complement components was observed in post-mortem brain samples from individuals with ASD, little is known about the expression patterns of complement molecules in distinct cell types in the developing autistic brain. In the present study, we characterized the mRNA and protein expression profiles of a wide range of complement system components, receptors and regulators in induced pluripotent stem cell (iPSC)-derived neural progenitor cells, neurons and astrocytes of individuals with ASD and neurotypical controls, which constitute in vitro cellular models that recapitulate certain features of both human brain development and ASD pathophysiology. We observed that all the analyzed cell lines constitutively express several key complement molecules. Interestingly, using different quantification strategies, we found that complement C4 mRNA and protein are expressed in significantly lower levels by astrocytes derived from ASD individuals compared to control astrocytes. As astrocytes participate in synapse elimination, and diminished C4 levels have been linked to defective synaptic pruning, our findings may contribute to an increased understanding of the atypically enhanced brain connectivity in ASD.


Introduction
The complement system, which plays crucial roles in the innate defense against pathogens and damaged cells [1], has been increasingly implicated in brain development and plasticity [2,3]. All major cell types in the brain were shown to constitutively express at least some components, receptors and regulators of the three pathways of complement activation -classical, lectin and alternative -, which contribute to some key cellular processes in the developing brain, including neurogenesis [4], neuronal migration [5], and synapse pruning and remodeling [6][7][8][9]. Consequently, inappropriate expression or activation of the complement system might alter the establishment of precise neural networks in the brain during development, resulting in cognitive dysfunction. Indeed, a growing number of studies has associated dysregulation of speci c components of the complement system with neurodevelopmental disorders [10], such as schizophrenia [8] and autism spectrum disorder (ASD) [11,12].
ASD is a heterogeneous neurodevelopmental condition characterized by de cits in social interaction and the presence of repetitive, restricted behaviors [13]. The etiology of ASD is very complex and may involve genetic, epigenetic, environmental and immunological risk factors [14,15]. Previous studies have found a potential association between abnormal expression of complement system elements and ASD risk.
Genetic variants in the complement C4B gene resulting in non-expression of the gene (C4B null alleles) were associated with increased risk for this disorder [16][17][18]. Altered levels of complement proteins -such as C4, C1q, C3, C5 and CFI -were observed in the periphery of individuals with ASD [19][20][21][22]. Finally, the expression patterns of several complement genes -such as C1q, C2, C3, C4, MASP1 and CR3 -were found to differ in post-mortem brain tissue samples from ASD subjects compared to control subjects [11,12].
However, the expression pro le of complement genes and proteins during the initial stages of human neurodevelopment and the speci c cell types in the brain from ASD individuals that differentially express complement components are still largely unexplored. Human induced pluripotent stem cell (iPSC)-derived brain cells have been successfully used to model ASD (23)(24)(25)(26), and a recent study from our group has shown that the transcriptome pro les from iPSC-derived neural progenitor cells (NPCs) and neurons best re ects neuronal tissue at early (4-10 post-conception weeks) and middle (16-24 post-conception weeks) stages of prenatal brain development respectively [27]. This study aimed to characterize the constitutive mRNA and protein expression levels of a wide range of complement system components, receptors and regulators in neuronal (NPCs and neurons) and glial (astrocytes) cells derived from iPSCs of individuals with ASD and neurotypical controls, and to identify possible differences in complement expression between the ASD and control cells.

Subjects and genetic analysis
All individuals evaluated in this study (n = 7 individuals with non-syndromic ASD and n = 5 neurotypical controls) were described previously [27][28][29][30]. CGH-array and whole exome sequencing using genomic DNA from peripheral blood of the ASD subjects and their parents allowed the identi cation of known ASD pathogenic variants in two individuals: one patient harbors deleterious compound heterozygous variants in the RELN gene, and a de novo splice site variant in the CACNA1H gene; the second patient harbors a duplication of 15q11-13. The remaining ve patients did not harbor rare variants that cause a known deleterious loss of function of an ASD gene.
Human iPSCs generation and differentiation into neural progenitor cells All iPSC samples used in this study have been previously generated and characterized [27,29]. For iPSC differentiation into NPCs, iPSCs were suspended to generate embryoid bodies and plated to produce neural rosettes, which were manually isolated, dissociated, and cultured on dishes coated with 10 µg/ml poly-L-ornithine and 5 µg/ml laminin in NPC medium containing: DMEM/F12, 0.25x N2-supplement (Thermo Fischer Scienti c), 0.5x B27-supplement (Thermo Fischer Scienti c), 20 ng/ml of FGF and 20 ng/ml EGF (Peptrotech, NJ, USA). Cell culture medium was changed every other day. NPCs derived from either 1 or 2 iPSC clones of each subject were used in all experiments described herein.

Differentiation of neural progenitor cells into neurons
Neurons were obtained after 4 weeks of differentiation from NPCs as previously described [27]. Brie y, NPCs were seeded on plates coated with 20 µg/ml poly-L-ornithine and 10 µg/ml laminin. After reaching 50% con uence, cells were cultured with DMEM/F12 medium supplemented with 0.5x N2, 1x B27, and 1 µM of retinoic acid (Sigma-Aldrich), which was changed every three days. Neurons derived from either 1 or 2 iPSC clones of each subject were used in all experiments described herein.

Differentiation of neural progenitor cells into astrocytes
Astrocytes were differentiated from NPCs as previously described [31]. Brie y, NPCs were mechanically detached and cultured in suspension in ultra-low attachment plates with NPC medium under agitation at 90 rpm to allow for 3D sphere formation. Twenty-four hours later, the medium was changed to DMEM/F12 supplemented with 0.5x N2, 0.5x B27 and 10 µM ROCK inhibitor, and spheres were maintained in suspension culture for 48 hours. Subsequently, the medium was changed to Astrocyte Growth Medium (AGM Bullet Kit, Lonza), and spheres were maintained in suspension culture for additional 15 days. Cell culture medium was changed every other day. The spheres were then seeded on plates coated with 10 µg/ml poly-L-ornithine and 5 µg/ml laminin and cultured in AGM until astrocytes spread from adherent spheres. When astrocytes reached con uency, spheres were manually removed and astrocytes were passaged at least three times before usage in any experiment. Astrocytes derived from either 1 or 2 iPSC clones of each subject were used in all experiments described herein.
Nuclei were stained with DAPI (VECTASHIELD, Vector Laboratories). Fluorescence images were obtained using a Zeiss LSM 710 confocal microscope system.

RNA extraction and quantitative real-time PCR
Total RNA from each cell line was extracted using the NucleoSpin ® RNA kit (Macherey-Nagel) following the manufacturer's instructions. RNA quality and quantity were assessed using the NanoDrop 3300 Fluorospectrometer (Thermo Fisher Scienti c). The cDNA was synthesized from 500 ng of total RNA with Multiplex and ELISA assays Cells were cultured until 90% con uence in 60 mm tissue culture plates, and the supernatants were collected after 48h cultures and centrifuged to remove cell debris. Cell-free supernatants (2.5 mL) were then transferred to a new conical tube, and the proteins in the supernatants were precipitated by adding ice-cold acetone (4:1) and resuspended in 250 ul of RIPA buffer containing protease inhibitors (10x concentrated supernatants). The concentration of 13 complement components and regulators were quanti ed in cell culture supernatants by using the Luminex xMAP detection system and the Human Complement Panel 1 and 2 Bead-Based Multiplex Assay kits (HCMP1MAG-19K and HCMP2MAG-19K, Millipore), whose detection sensitivities are (minimum detectable concentrations): C2 = 310 pg/ml, C4b = 360 pg/ml, C5 = 1040 pg/ml, C5a = 5.1 pg/ml, CFD = 26pg/ml, MBL = 40 pg/ml, CFI = 220 pg/ml, and C1q = 48 pg/ml, C3 = 120 pg/ml, C3b/iC3b = 3639 pg/ml, C4 = 191 pg/ml, CFB = 24 pg/ml, CFH = 135 pg/ml respectively. Data acquisition and analysis were performed using the Luminex ® xPONENT ® and the Milliplex ® Analyst 5.1 softwares (Millipore), respectively. Values below the minimum detectable concentrations of the multiplex assays were not considered. C4 protein levels were also quanti ed in astrocyte culture supernatants in a full set of independent experiments (including novel astrocyte differentiation, protein extraction and quanti cation) by using an ELISA assay (Human Complement C4 ELISA kit, ab108825, Abcam) following manufacturer's instructions. Optical densities measurements were taken at 450 nm using a GloMax® Discove Microplate Reader (Promega). All test samples fell above the minimum detectable concentration of the ELISA assay (41 pg/ml). Results were normalized to the total protein content of the cell lysates and were expressed in pg/ml. The dot-plots show the protein levels for each individual in the Control and ASD groups and combine results from two independent experiments.

Statistical analysis
Statistical analyses were carried out using the generalized linear mixed-effect model in SPSS software to account for dependency between biological replicates (when NPCs, neurons and astrocytes derived from different iPSC clones of the same individual were used) and independent technical replicates per individual. P values < 0.05 were considered statistically signi cant. Data obtained by RT-qPCR, multiplex and ELISA analysis were represented as median with interquartile range.

Results
Generation of iPSC-derived neural progenitor cells, neurons and astrocytes from ASD and control subjects iPSCs from individuals with ASD (n = 7) and control subjects (n = 5) were differentiated into NPCs, postmitotic neurons and astrocytes, and the expression of lineage-speci c markers was con rmed by immunocytochemistry and western blotting. We did not detect any major differences between ASD and control cells with respect to the differentiation capacities and no signi cant differences in the expression levels of the lineage markers were observed between the two groups (Fig. 1). To examine the constitutive expression pro les of complement system in these human neuronal and glial cell types, we combined mRNA and protein quanti cation assays.

Expression of complement genes in iPSC-derived neural progenitor cells, neurons and astrocytes
First, we examined the gene expression levels of several complement system components (C1R, C1S, C2, C3, C4A/B, C5, C7, C9, MBL2, MASP1, MASP2, CFB, CFD), receptors (C3aR1 and C5aR1), and regulators (SERPING1, CFH, CFI, and CD59) in control-and ASD-derived NPCs, neurons and astrocytes by RT-qPCR. The results showed that, except for MBL2, mRNA transcripts for these key complement genes are detected in all cell samples analyzed. Some genes exhibit highly variable expression across samples, and the expression values of most genes are signi cantly higher in neurons than in NPCs and astrocytes (Fig. 2a-c). No signi cant differences in mRNA expression levels were found between control and ASD NPCs, as well as between control and ASD neurons. On the other hand, a signi cant decrease in the expression levels of C4A/B (paralogous C4A and C4B genes) was observed in astrocytes derived from ASD subjects compared to control astrocytes (p = 0.009) (Fig. 2c).
Expression of complement proteins in iPSC-derived neural progenitor cells, neurons and astrocytes Next, protein levels of several soluble complement components (C1q, C2, C3, C3b/iC3b, C4, C4b, MBL, C5, C5a, CFB, CFD) and regulators (CFH and CFI) in culture supernatants from control-and ASD-derived NPCs, neurons and astrocytes were measured by multiplex assays. The results indicated that all cell samples secrete reliably detectable amounts (above the lower limit of detection of the multiplex assays) of C4, C5a, CFB, CFH and CFI, and that the expression levels of most of these proteins vary substantially across samples. Notably, neurons and astrocytes secreted signi cantly higher levels of C4 than NPCs, and detectable levels of C4b, a C4 cleavage product, were only observed in the supernatants of these cell types. Also, astrocytes and neurons also secrete reliably detectable levels of CFD (Fig. 3a-c). Protein levels of C1q, C2, C3, C3b/iC3b, C5, and MBL were below the lower limit of detection of the multiplex assays in culture supernatants from most samples and were not considered for further analysis (data not shown).
Interestingly, whereas no signi cant differences in the secretion levels of complement proteins were found between control and ASD neuronal and glial cells, we observed a strong tendency towards decreased secretion of C4 and C4b by ASD astrocytes compared to control astrocytes (Fig. 3c), which is in accordance with the signi cant diminished C4A/B mRNA expression in these cells. In order to con rm the obtained results, the C4 protein level was also quanti ed in astrocyte culture supernatants by using a human C4 ELISA assay and a full set of independent experiments (with a larger sample of astrocytes). The results revealed that ASD astrocytes under steady-state conditions indeed secret signi cantly lower levels of C4 compared to control astrocytes (p = 0.001) (Fig. 3d).

Discussion
In this study, we investigated the production of the main components of the three complement pathways and their key receptors and regulators in human iPSC-derived NPCs, neurons and astrocytes of ASD and control subjects, which are in vitro models that recapitulate some molecular and cellular aspects of the developing human brain.
We observed that although a large number of complement genes are transcribed in human NPCs, neurons, and astrocytes, these cells under normal conditions may be an effective local source of only a speci c set of complement components and regulators. We found that all analyzed cell samples express mRNA transcripts for C5 and C5a receptor (C5aR1), and secrete similar levels of the C5a protein, produced from cleavage of C5. Studies using mouse models have shown that absence of C5a-C5aR1 signaling results in neural tube defects in folate-de cient mouse embryos [32], and that pharmacological blockade of C5aR1 during neurogenesis inhibits NPC proliferation in the ventricular zone of mouse embryos and leads to behavioral abnormalities later in life [4]. Interestingly, in vitro studies have shown that C5a and C5aR1 are constitutively expressed in human embryonic stem cells and iPSCs regulating their pluripotency [33], in human NPCs promoting polarization and proliferation of these cells [34], in human fetal astrocytes regulating calcium transient activity [35], and also in mouse cortical neurons inducing apoptosis [36,37]. Although we did not observe any differences in the expression patterns of C5a and C5aR1 between ASD and control neuronal and glial cells, our results corroborate the ndings of previous studies showing expression and physiological roles of these complement molecules in different cell types in the developing human brain.
We also found that all analyzed ASD and control cell lines express CFB mRNA and secrete relatively low levels of CFB protein, an activator of the alternative pathway. In addition, although all cell lines express CFD mRNA, CFD protein, a co-factor of CFB that activates the alternative pathway, was only reliably detected in neuron and astrocyte supernatants. We also observed that all ASD and control cell lines analyzed express CFH and CFI mRNA and secrete relatively high levels of CFH and CFI proteins, which are inhibitors of the classic and alternative pathways of the complement system. Whereas the expression of these complement components and regulators are implicated in cerebral in ammation [38], degeneration [39] and retinopathies [40,41], the physiological roles of these proteins in the developing human nervous system deserve further investigation.
Finally, we observed constitutive expression and secretion of the critical component of both classical and lectin cascades C4 by ASD and control NPCs, neurons and astrocytes, and also constitutive secretion of C4b by ASD and control neurons and astrocytes. Interestingly, we found that astrocytes derived from individuals with ASD express signi cantly lower levels of C4A/B mRNA and secret signi cantly lower levels of C4 protein compared to control astrocytes. In the brain, C4 localizes to synapses and, together with other members of the classic complement cascade, has been shown to be required for synaptic elimination by microglia in the mouse developing visual system, as mice de cient for complement C1q, C3, CR3 or C4 exhibit impaired elimination of retinogeniculate synapses [6][7][8]. Accordingly, overexpression of C4 in the mouse prefrontal cortex caused alterations in dendritic spine development, reduced connectivity, increased synaptic pruning, and de cits in social behavior [9].
In humans, C4 is encoded by two different genes, C4A and C4B, which are located in tandem on the short arm of chromosome 6 and vary in copy number [8]. Whereas C4B null alleles that decrease the expression of C4 have been associated with ASD [16][17][18], alleles at C4A that increase the expression of C4 have been associated with schizophrenia and, mechanistically, it has been proposed that augmented C4 is involved in the exacerbated synaptic pruning and decreased synapse number in schizophrenic patients [8]. Therefore, taking all the above mentioned data into account and the fact that astrocytes regulate synapse formation, elimination and activity [42][43][44], it is tempting to speculate that a reduced secretion of astrocyte-derived C4 might contribute to the reduced synaptic pruning and increased dendritic spine density in the brain of individuals with ASD [45,46]. However, additional studies are clearly needed to determine the consequences of decreased astrocyte-derived C4 in brain development and in ASD pathophysiology, and also to explore the C4 locus structure in ASD individuals.
In summary, our results provide insights into the expression patterns of a broad range complement genes and proteins in iPSC-derived NPCs, neurons and astrocytes, and revealed decreased expression and secretion of complement C4 by astrocytes derived from ASD individuals. Also, our ndings highlight the use of human iPSC-derived neuronal and glial cells as effective platforms for the study of the complement system in human neurodevelopment.