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 specific 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-deficient mouse embryos , 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 . Interestingly, in vitro studies have shown that C5a and C5aR1 are constitutively expressed in human embryonic stem cells and iPSCs regulating their pluripotency , in human NPCs promoting polarization and proliferation of these cells , in human fetal astrocytes regulating calcium transient activity , 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 findings 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 inflammation , degeneration  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 significantly lower levels of C4A/B mRNA and secret significantly 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 deficient for complement C1q, C3, CR3 or C4 exhibit impaired elimination of retinogeniculate synapses [6–8]. Accordingly, over-expression of C4 in the mouse prefrontal cortex caused alterations in dendritic spine development, reduced connectivity, increased synaptic pruning, and deficits in social behavior .
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 . Whereas C4B null alleles that decrease the expression of C4 have been associated with ASD [16–18], 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 . Therefore, taking all the above mentioned data into account and the fact that astrocytes regulate synapse formation, elimination and activity [42–44], 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 findings highlight the use of human iPSC-derived neuronal and glial cells as effective platforms for the study of the complement system in human neurodevelopment.