C1q drives neural stem cell quiescence by regulating cell cycle and metabolism through BAI1

Domain: physics
Published: 2025-12-11
Editorial outcome: major_revision

Abstract
C1q levels in the CNS are elevated by inflammation and neurovascular trauma, yet the consequence of this increase for neural stem cell (NSC) regeneration response remain unknown. We have recently identified C1q receptor candidates that regulate NSC behavior. One of these is Brain Angiogenesis Inhibitor 1 (BAI1, ADGRB1), which has no previously discovered role in NSC. Here, we show that C1q acts in a BAI1-dependent manner to modulate NSC quiescence via two parallel mechanisms. First, negative regulation of MDM2, driving cell cycle suppression through p53. Second, endocytic internalization of C1q-BAI1-complex, driving regulation of p32 (C1qBP) and metabolic reprogramming towards aerobic glycolysis. We validated the biological significance of BAI1 in a male hNSC line in vivo using a female mouse model of acute spinal cord injury (SCI). These findings are relevant for a multiplicity of CNS disorders, and illuminate complex connections between C1q, cell cycle, and metabolism. Together, these data provide valuable insight into C1q-mediated regulation of NSC transition between activation and quiescence, processes fundamental for tissue development and repair.

Introduction
Adult tissue-specific stem cells maintain tissue homeostasis by shifting from quiescence to activation. Strict regulation over cell activation, cell cycle progression, and lineage-specific differentiation is crucial to prevent stem cell pool exhaustion1. During aging, tissues exhibit progressive declines in stem cell activation, contributing to loss of regenerative capacity and pathology2. This is exacerbated in the CNS, where regenerative capacity is already limited relative to other tissues3–5. Additionally, CNS aging, neurodegenerative disease, and injury are associated with prolonged inflammation6–9, including exposure of the NSC-neurovascular niche to complement C1q derived from microglia10,11 and blood-brain/spinal cord-barrier (BBB/BSB) dysfunction12,13. This results in a 400-fold increase in the accumulation of C1q in the aged brain6. Similarly, C1q levels in peripheral serum increase almost twofold during aging14. Age-related increases in C1q induce declines in skeletal muscle regeneration via direct effect on satellite stem cell proliferation15, however, the effect of C1q on declines in NSC activation (proliferation) vs. maintenance (self-renewal) has not been investigated.

Mitochondria and metabolic rewiring have emerged as central regulators of activation and maintenance of the NSC pool16,17, and both CNS aging and neurodegenerative disease are linked to impairments in energy metabolism and metabolic dysfunction. Although complement proteins regulate immune cell metabolism and function via the complosome18,19, a direct role or mechanism of action for C1q in neural metabolism has not been established.

We recently identified C1q candidate cell surface receptors in NSC20, enabling the application of gene editing to study the role of C1q in NSC function. Here, we demonstrate that blood plasma C1q concentrations associated with aging and BBB/BSB breakdown drive declines in NSC proliferation in vitro and in vivo in a CNS trauma model. We identify that the underlying mechanism is dependent on an interaction with BAI1, and distinct from that shown for muscle satellite cells, driving not only cell cycle arrest but also metabolic changes associated with stem cell quiescence.

Results
C1q promotes hNSC quiescence
To investigate the effect of C1q on hNSC proliferation, we exposed hNSC to purified human C1q at physiological concentrations ranging from 0.1 nM to 300 nM, followed by a BrdU incorporation assay. As reported for muscle satellite cells, C1q concentrations associated with circulating blood plasma [100 nM-300 nM] dramatically decreased the proliferating hNSC population (Fig. 1a, b). hNSC survival (Real Time-Glo Annexin V time course analysis; Fig. 1c, d) and apoptosis (cleaved caspase 3, CC3; Supplementary Fig. 1a, b) were unaltered in C1q-treated hNSC vs. non-treated controls. In addition, C1q-treated hNSC exhibited a rebound in proliferation when returned to normal in vitro growth conditions (Fig. 1e), demonstrating reversibility of this effect. The effect of C1q on hNSC proliferation was blocked by co-treatment with a previously characterized C1q-neutralizing antibody but not an IgG control (nAb; Fig. 1f)5,20.

We tested the role of cell cycle modulation in C1q-driven proliferation decline using Fucci-mNSC (Fig. 1g–i, Supplementary Fig. 1c)21–23. Live-cell time-lapse and quantification of cell cycle dynamics revealed that C1q [200 nM] promoted G0/G1 phase arrest and decreased progression to G1/S or S/G2/M vs. non-treated controls (Fig. 1h, i). Consistent with Real Time-Glo Annexin V analysis, C1q-driven G0/G1 arrest increased cell survival rather than cell death in Fucci-mNSC (Fig. 1j and Supplementary Fig. 1d, e). These data identify that exogenous C1q negatively regulates cell cycle progression/proliferation in NSC. A reversible loss of proliferation combined with cell cycle arrest at G0 in the absence of cell death (Fig. 1a–j) is consistent with stem cell quiescence24,25, which would predict a decrease in self-renewal. As hypothesized, clonal neurosphere assays revealed a significant decrease in hNSC self-renewal during C1q [200 nM] exposure vs. non-treated controls (Fig. 1k, l). No significant change in neurosphere size was detected during C1q [200 nM] treatment vs. non-treated controls (Supplementary Fig. 1f). Additionally, stem cell quiescence is distinct from cell cycle arrest during terminal differentiation. Analysis of total protein lysates from hNSC exposed to C1q [200 nM] during in vitro differentiation revealed significant increases in undifferentiated NSC/progenitor markers, nestin and GFAPδ26, but not lineage commitment markers (Fig. 1m). RNA seq further identified significant transcriptomic enrichment of quiescence genes in C1q [200 nM] treated hNSC (Fig. 1n, grey), shown by radar plots identifying enriched pathways (labels) and magnitude of change (distance from center). These data indicate that C1q induces quiescence in hNSC.

While the effect of C1q on NSC proliferation was reversible after short-term exposure (up to 48 h, Fig. 1e), CNS injury/aging can cause prolonged C1q elevation. We therefore tested whether long-term exposure to C1q has a priming effect on hNSC proliferation or self-renewal. hNSC neurosphere cultures were exposed to C1q for 2, 4, 6, or 8 weeks, at which time C1q was withdrawn and cells returned to normal in vitro growth conditions for analysis 5 weeks later. Consistent with short-term exposure effects (Fig. 1e), hNSC proliferation declines were reversed after C1q withdrawal (Fig. 1o). hNSC self-renewal declines (Fig. 1l), however, persisted after long-term priming (Fig. 1p), and were exacerbated by the length of exposure. Long-term C1q [200 nM] priming caused no significant differences in neurosphere size relative to non-treated controls (Supplementary Fig. 1g), supporting the interpretation that there were fewer neurosphere-initiating cells present in the C1q [200 nM] primed group over time. Apoptotic cell death again remained unchanged (Supplementary Fig. 1h, i), and hNSC retained multi-lineage differentiation capacity (Supplementary Fig. 1j). Depending on factors within the niche24,25, quiescence exit of NSC can occur through any of the following modes of cell divisions: self-renewing symmetric, in which two new NSC; self-renewing asymmetric are produced, in which one of the two cells remains as an NSC and another initiates terminal differentiation; or self-consuming symmetric, in which two faster proliferating transit amplifying (TA) progenitor that both initiate terminal differentiation are produced27. The self-consuming symmetric cell divisions leading to terminal differentiation can thus deplete the originating stem cell population and result in stem cell exhaustion. Therefore, the observed long-term C1q priming induced proliferation rebound and increase, coupled with an overall decline in self-renewal, could be interpreted as NSC shifting their mode of division from self-renewing to self-consuming. This would suggest that extended exposure to C1q at blood plasma concentrations could affect maintenance of the NSC pool and long-term tissue turnover, providing an important insight into how C1q could contribute to CNS pathological processes and impact repair capacity. Critically, decline of the neural stem cell pool is posited to be a major factor in the age-related decline of regenerative functions in the CNS, and is thought to derive from increased quiescence and terminal differentiation24,28.

C1q-driven proliferation decline has been previously reported in muscle satellite cells, where C1q in C1 complex binds to Frizzled receptor, resulting in C1s-mediated cleavage of LPR6 and Wnt pathway activation15. To investigate whether C1q modulates hNSC proliferation through the same mechanism, we evaluated expression of these transcripts. hNSC expressed Wnt ligands, Frizzed receptors, LRP, including LRP5/6, and C1s/C1r, but not C1q or downstream complement pathway components required for formation of the membrane attack complex (Supplementary Table 1). Despite the absence of C1q transcripts, paracrine/exogenous C1q could theoretically combine with autocrine C1r/C1s to form the C1 complex. However, in contrast to muscle satellite cells, neither a C1s inhibitor (Fig. 1q) nor a C1 complex-inhibitor (Fig. 1r) rescued C1q-induced proliferation decline. Further, purified human C1 complex had no effect on hNSC proliferation (Fig. 1s). These data point to a different mechanism for the action of C1q on NSC, in which C1q selectively acts extracellularly as a single ligand.

C1q modulates p53 activity towards quiescence
C1q has been shown to drive activation of specific intracellular signaling pathways in a concentration-dependent manner in hNSC20. We searched these data for an intracellular signaling signature that could provide mechanistic insight, identifying that C1q [200 nM] uniquely increases phosphorylation of p53 serine 15 (p53Ser15) (Fig. 2a). We confirmed this observation by Western blot vs. non-treated and temperature-inactivated C1q controls (TInact; Fig. 2b). p53 is a critical regulator of stem cell proliferation and self-renewal29, and p53Ser15 is important for protein stability and transcriptional activation, particularly of the cell cycle arrest protein p2130,31. Nuclear p53 functions as a transcription factor; however, mitochondria/cytoplasmic p53 is linked to other cellular functions32–34. We analyzed the effect of C1q on phospho-p53Ser15 subcellular localization, identifying a selective increase in nuclear localization in C1q [200 nM]-treated hNSC vs. controls (Fig. 2c, d). Consistent with nuclear p53 localization, genes associated with p53 downstream activation (Fig. 2e, f) and cell cycle arrest (Fig. 2g) were significantly enriched and differentially expressed in C1q [200 nM]-treated vs. control hNSC in RNAseq. These data identify p53 as a possible driver for the C1q effect on NSC cell cycle.

C1q-driven increase in p53 signal transduction is BAI1-dependent
p53 sub-cellular localization and function are modulated by the MDM2 E3 ubiquitin ligase35. In medulloblastoma, the G-protein-coupled receptor BAI1 binds MDM2, protecting p53 from degradation and suppressing tumor cell proliferation36. We recently identified C1q as a ligand for BAI1 in NSC20. While BAI1 engagement with its ligands has been shown to modulate intracellular signaling37,38, whether BAI1 engagement with ligands such as C1q can alter BAI1-MDM2 binding to modulate protein degradation is unknown. We hypothesized that C1q-BAI1 engagement could increase BAI1-MDM2 binding as a mechanism for C1q-driven increases in p53 phosphorylation and nuclear translocation (Fig. 3a). We generated a BAI1 knockout (KO) hNSC line to test this hypothesis. BAI1 KO cells retained normal characteristics, including karyotype, CD133+ stem cell proportion, stable growth rate under in vitro growth conditions, migration response, and multipotency in neural lineage differentiation (Supplementary Fig. 2). We validated loss of BAI1 in KO hNSC by Western blot (Fig. 3b), as well as loss of C1q-BAI1 interaction in these cells by proximity ligation assay (PLA; Fig. 3c, d) using commercial BAI1 antibodies tested in BAI1 HEK293T overexpression vs. negative control lysates (Supplementary Fig. 3a). We investigated whether C1q altered BAI1-MDM2 binding in wild type (WT) hNSC by PLA, identifying a strongly significant increase in response to C1q that was abolished in BAI1 KO hNSC (Fig. 3e, f). PLA specificity was validated using a mismatched (negative) interaction control (MET-MDM2; Fig. 3g, h). We also evaluated the dependence of C1q-mediated changes on p53 phosphorylation on BAI1. We replicated the C1q-mediated increase in p53 normalized total p53Ser15 (Fig. 3i) and shift in p53Ser15 localization to the nucleus (Fig. 3j, k) in BAI1 WT hNSC, and confirmed that these effects were reversed by BAI1 KO. These data demonstrate that C1q-BAI1 interaction promotes BAI1-MDM2 binding and p53 activity.

Critically, BAI family proteins have other known roles in cellular function, suggesting that MDM2/p53 signaling may not be the only mechanism modulating hNSC quiescence. Ligand-receptor complex internalization by endocytosis is key to GPCR modulation of intracellular signaling39,40. Both BAI1 and BAI3 GPCR family members have identified roles in endocytosis41–46. In parallel, intracellular complement is emerging as an orchestrator of cellular functions in immune cells47; however, only two reports have described intracellular C1q in neural lineage cells48,49, and no intracellular role for C1q has been established in CNS cells. We therefore hypothesized that C1q could be internalized in complex with BAI1 via endocytosis, modulating the proliferative state of hNSC intracellularly through a second mechanism.

BAI1-C1q endocytosis decreases total p32
C1q internalization kinetics were analyzed using single-cell Imagestream multispectral imaging flow cytometry50. C1q immunolabeling was detected in association with 46% of in-focus single cells in C1q [200 nM]-treated cultures (Fig. 4a); component masking analysis51 of this subpopulation identified C1q internalization within 5 min, increasing through 24 h post-treatment (Fig. 4b, c, e, Supplementary Figs. 3b, 4m). Internalized C1q colocalized with early endosomes (Fig. 4d, f), but not with Golgi (Fig. 4g). We further verified C1q internalization in hNSC using immunogold electron microscopy (Fig. 4h).

We investigated the intracellular proteins/pathways with which internalized C1q interacts via an unbiased forward screen using nanoLC-MS/MS, and a pull-down approach with a cell-permeable cross-linker in hNSC monolayer cultures. While this approach limits sensitivity, the 3D structure of C1q-bait and interacting intracellular prey-proteins is maintained, maximizing biological relevance. We detected a total of 214 prey proteins that were significantly different in C1q vs. non-treated samples. Subcellular enrichment analysis identified a significant association of these proteins with several specific compartments involved in receptor-mediated endocytic trafficking of C1q (Fig. 4i, Supplementary Table 2), including the cytoskeletal, cytoplasmic, vesicular, lysosome, ribosome and endoplasmic reticular compartments. Subcellular interactions were specific, as no enrichment was detected in the mitochondria or peroxisome, which are not linked to endocytic pathways of protein internalization. Further supporting specificity, the cell-permeant cross-linker paradigm did not detect enrichment at the cell membrane.

We next determined whether C1q is internalized in complex with BAI1. Imagestream analysis was validated using two matched positive controls (Fig. 4j and Supplementary Fig. 4a–i), and a mismatched negative control (Fig. 4k)52,53. In C1q [200 nM]-treated cultures, BAI1 was detected in 78% of in-focus single cells (Fig. 4l, n), and intracellular C1q-BAI1 co-localization was identified within 5 min (Fig. 4o, Supplementary Fig. 3c, Supplementary Fig. 4j, k). BAI1 KO decreased both the proportion of in-focus single cells with intracellular C1q (Fig. 4p and Supplementary Fig. 4l) and the overall quantity of internalized C1q per cell within this sub-population (Fig. 4q and Supplementary Fig. 4n)54. These data suggest that C1q-BAI1 engagement not only modulates p53 signaling but also serves as an endocytic portal for C1q in NSC.

In T cells, C1q interaction with cell surface p32 results in C1q endocytosis followed by C1q-p32 transport to the mitochondria via an unknown mechanism55. In contrast to T-cells, no evidence of cell surface p32 was detected in hNSC by either Imagestream (Supplementary Fig. 4o) or protein enrichment of specific cellular compartments (Fig. 4r, s). Rather, p32 was localized intracellularly, particularly in the mitochondrial matrix (Supplementary Fig. 4p), consistent with a well-established role for p32 in mitochondrial protein translation and OXPHOS activity56,57. We therefore hypothesized that BAI1-mediated C1q endocytosis would enable intracellular C1q-p32 interaction. In C1q [200 nM]-treated cultures, p32 was detected in 82% of in-focus single hNSC (Fig. 4m, t), and intracellular C1q-p32 co-localization was identified within 30 min (Fig. 4u). Intracellular C1q-p32 interaction was confirmed by PLA (Fig. 4v, w). Because Parkin and ArgII have been shown to regulate p32 stability58, we hypothesized that C1q might do the same. Consistent with this idea, p32 protein was significantly decreased post-C1q treatment (Fig. 4x). This decrease was dependent on BAI1, as shown by Imagestream (Fig. 5a, b), PLA (Fig. 5c, d), total p32 protein analysis (Fig. 5e), and mitochondrial p32 localization (Supplementary Fig. 5c–e) in BAI1 KO vs. WT cells. Critically, p32 transcription was unaltered by C1q treatment in WT cells, and p32 protein levels were unaltered in non-treated (baseline) WT vs. KO cells (Supplementary Fig. 5a, b). His-tag pull-down of purified human proteins in situ demonstrated a direct interaction between C1q-BAI1 and C1q-p32, as well as BAI1-p32, and that C1q-BAI1-p32 can be pulled down as a complex (Fig. 5f). Together, these data show that BAI1-mediates endocytosis of C1q enabling intracellular protein interactions between C1q-p32, BAI1-p32, or C1q-BAI1-p32, decreasing total p32.

C1q drives aerobic glycolysis via BAI1
p32 participates in several cellular functions, however, its most prominent role is to regulate mitochondrial metabolism. p32 cleavage or KO drives a shift from OXPHOS to glycolysis as well as loss of proliferation56,57,59,60. We therefore hypothesized that C1q-treated hNSC would exhibit a BAI1-dependent C1q-mediated glycolytic shift in cell metabolism (Fig. 5g) consistent with NSC quiescence61–63. We tested whether C1q treatment induces a glycolytic shift in hNSC, evaluating gene expression profile and NAD/NADPH ratio. RNA seq analysis followed by Gene Set Enrichment Analysis (GSEA) revealed a significant upregulation of glycolysis transcripts in C1q [200 nM] vs. control hNSC 48 h post-treatment (Fig. 5h, i). In parallel, timecourse analysis of NADH/NAD+ redox ratio using phasor two-photon fluorescence lifetime imaging microscopy (FLIM) identified a glycolytic shift after C1q [200 nM] treatment (Fig. 5j, k, Supplementary Fig. 6a)64–66. Time-course analyses of mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) generation indicated that this outcome was not associated with mitochondrial dysfunction detectable as a loss of Δψm or broad increase in ROS (Supplementary Fig. 6b, c). Thus, C1q [200 nM] treatment shifts hNSC metabolism from OXPHOS to aerobic glycolysis.

We next investigated the effect of BAI1 KO on hNSC mitochondria fission, which is associated with both p32 and increased glucose metabolism59,67, as well as on Seahorse metabolic analysis. Non-treated BAI1 WT and KO hNSC exhibited comparable baseline parameters (Supplementary Fig. 7a–c), however, a small shift in bioenergetics was detected in BAI1 KO hNSC (Supplementary Fig. 7c); data was therefore normalized to non-treated controls within each line. C1q [200 nM]-treated BAI1 WT hNSC exhibited increased mitochondrial fission (Fig. 5l, m), decreased oxygen consumption rate (OCR) (Fig. 5n), and increased extracellular acidification rate (ECAR) (Fig. 5o) vs. non-treated controls, each of which was reversed by BAI1 KO. Neither C1q treatment nor BAI1 KO affected hNSC ROS (Supplementary Fig. 7a), H+ proton leak, coupling efficiency, or non-mitochondrial oxygen consumption (Supplementary Data Fig. 7d), suggesting a specific effect on mitochondrial energy metabolism. These data provide evidence that C1q directly influences neural cell lineage bioenergetics, revealing a BAI1-dependent mechanism for these effects.

BAI1 KO rescues C1q-induced proliferation decline in hNSC
In all, these data identify that C1q drives a decline in hNSC proliferation caused by reversible G0/G1 cell cycle arrest that is consistent with quiescence, and is mediated by at least two parallel mechanisms involving MDM2-p53 and p32. To confirm the relevance of BAI1 to cellular function we used proliferation and self-renewal as outcome measures. In vitro blockade of BAI1 using a characterized BAI1-neutralizing antibody (R&D systems, AF4969), as well as BAI1 KO, reversed C1q-mediated hNSC proliferation decline (Fig. 6a, b). Consistent with these results, BAI1 KO hNSC exhibited a dramatic reversal of C1q-driven loss of cell divisions that maintain the stem cell pool (Fig. 6c), suggesting restoration of a self-renewing mode of division.

We next sought to validate the biological significance of BAI1 in hNSC in vivo, selecting acute spinal cord injury (SCI) as the study paradigm. C1q increases after CNS trauma such as SCI68,69. We analyzed local C1q expression by different cell types using published scRNAseq data70 as well as total protein in our injury model. Nearly all immune cell populations and spinal cord cell types upregulated C1qa mRNA expression by 3–7 days post-injury (dpi; Fig. 7a, b), with C1q protein peaking in the SCI epicenter 3–24 hours post-injury (Fig. 7c), suggesting that both BSB disruption and local cell populations contribute to bioavailable C1q after SCI. We have also demonstrated that blockade of C1q at the lesion epicenter modulates the recruitment of transplanted hNSC after acute SCI5,20, we therefore predicted that BAI1 KO would increase early hNSC engraftment. BAI1 WT and KO hNSC were transplanted into an acute SCI model, and proliferating hNSC were identified by co-labeling for STEM121 and BrdU at 2 weeks post-transplantation. Stereological analysis revealed a significantly greater number of BAI1 KO vs. WT hNSC in this paradigm (Fig. 7d–f). BAI1 KO did not modulate migration in vitro or in vivo (Supplementary Fig. 2j and Supplementary Fig. 7e, f). These data show that BAI1 KO rescues proliferation of transplanted hNSC, consistent with a reduction in quiescence signaling, and supporting a biologically significant role for C1q and BAI1 in the negative regulation of NSC in the CNS.

Methods
Stem cell lines
Two multipotent human male fetal CNS-derived stem cell lines (hNSC; Stem Cells Inc. 2491.294 and UCI161, UCIrvine, Anderson AJ) were isolated from gestational week 16–20 fetal brain using comparable derivation methods and enriched for CD133 stem cell marker94,95. BAI1 WT and KO hNSC were generated using CRISPR-Cas9 editing of UCI191 hNSC (Supplementary Fig. 2). All hNSC lines displayed neurosphere formation, chemotactic responsiveness, and stable multipotency for >20 passages, generating neurons, oligodendrocytes, and astrocytes94,95. A multipotent mouse NSC line was derived from single Fucci mouse21 cortices at E11 (Fucci-mNSC) as previously described in ref. 23. Both human and mouse NSC showed reduced proliferation in response to C1q. Sex was not considered in all elements of in vitro analysis of human cells because of the volume of data collected. While sex-based differences between NSC lines were not performed in this study, both male and female hNSC lines express BAI1 and responded to C1q (Supplementary Fig. 7g, h).

Mice
Ten-week-old immunodeficient female Rag-1-mice (n=14; B6.129S7-Rag1tm1Mom/J; Jackson Laboratory, 002216) were used as the SCI model. Sex was not considered in the study design; Females were selected to minimize bladder complications and urolithiasis common in male mice following SCI96,97. Mice were housed in cages with Alpha-dri bedding, nestlets, and housing under IACUC approved conditions guidelines with a 12 h dark/light cycle and access to food and water ad libitum. Fucci reporter mice23,98 were bred to generate Fucci-mNSC line for live imaging of cell cycle dynamics.

Proliferation, neurosphere formation, and Real Time-Glo Annexin V multiplex assay
Human and mouse NSC were cultured as free-floating neurospheres in growth medium (GM) as previously described in ref. 94. BrdU (AmershamTM Cell Proliferation Kit, GE Healthcare, 45-000-837) and EdU proliferation assays (Invitrogen, C10338) were performed according to the manufacturer instructions in monolayer cultures on poly-L-ornithine (PLO) and laminin-coated chamber slides without (non-treated controls) or with purified human C1q (MyBioSource, MBS147305) or purified human C1 (CompTech, A098). BrdU or EdU was incorporated for 48 h before fixation (4% PFA) and immunostaining. Hoechst (1 µg/mL; Invitrogen, H3570) was used as a nuclear counterstain. For preconditioning, hNSC were exposed to 200 nM C1q for 48 hours (h), followed by washout and 48 h BrdU incorporation. Biologically independent experiments were performed with technical duplicate wells. Images were acquired using Olympus IX, Zeiss LSM900 or ApoTome, or Keyence BZ-X microscopes with 20x objective and analyzed using Imaris (Andor Technology, version 7.5.2. or 9.1.2) by investigators blinded to experimental conditions/groups. BrdU/EdU/Hoechst% per each captured image/independent experiment were tested for outliers using ROUT (Q=10%) in Prism (GraphPad, version 10); outliers are listed in the Source Data.

For clonal neurosphere assays, free-floating neurospheres were dissociated into single cells (Supplementary Methods) and plated into a 96-well plate (15 cells/well) in GM, with or without C1q. Sphere-forming units were calculated at 2.5 weeks (w) post-plating as the proportion of new neurospheres (⌀>50 µm) to plated cells99, and neurosphere sizes were measured using ImageJ (NIH). For C1q priming assays, free-floating neurospheres were co-cultured with or without C1q 48 h or for up to 6–8 w followed by wash out and plating as a monolayer for BrdU or a low adherence for neurosphere initiation in GM. Post-C1q wash, BrdU was incorporated for 48 h, and sphere forming units were analyzed 5 w later.

Real Time-Glo Annexin V (Promega, JA1011) apoptosis and necrosis assays were performed according to the manufacturer instructions with or without C1q. hNSC treated with 5 µM ionomycin (Sigma-Aldrich, I3909) or 0.8 µM stauroporine (Sigma-Aldrich, S6942) were used as positive controls. Biologically independent experiments were performed with technical triplicates. Quantification of all experiments were performed by investigators blinded to experimental conditions/groups. More details available in the Supplementary Methods.

C1q-, C1s- and BAI1 neutralizing antibodies and C1 inhibitor
hNSC were plated as monolayers on PLO/laminin-coated chamber slides with or without C1q and co-cultured with C1q nAb goat anti-human C1q (Quidel, A031) or normal goat IgG control (R&D systems, AB-108-C), complement component C1s antibody (Novus Biologicals, NBP1-52122) or mouse IgG control (Invitrogen, 1903), C1 inhibitor (Millipore, GF178), human BAI1 Antibody (R&D systems, AF4969) or normal sheep IgG control (R&D systems, 5-001-A) for 48 h in presence of BrdU or Edu. PFA 4%-fixed, immunostained, and imaged samples were analyzed using Imaris software by investigators blinded to experimental conditions/groups. Biologically independent experiments were performed with technical duplicate wells. BrdU/EdU/Hoechst% per each captured image/independent experiment were tested for outliers using ROUT (Q=10%) in Prism; outliers are listed in the Source Data.

Fluorescence live cell imaging and tracking
Time-lapse image acquisition of Fucci-mNSC monolayers cultured in custom-made PDMS without (non-treated control) or with 0.1 nM or 200 nM C1q and analysis were performed as previously described23. Each biologically independent experiment included 5 technical replicate microwells per group, one microwell per group with comparable cell confluences was captured and analyzed per each independent experiment. The total number of cells and proportions of G1 or S, G2, or M cell phases were quantified at 0 h, 48 h, 72 h, 96 h, and 7 days (d). For single-cell tracking of NSC survival, 30 Fucci-mNSC in G1 phase were randomly selected per biologically independent experiment and tracked up to 7 days using Imaris software as previously described in ref. 23. Cell death was calculated as the average percentage of dead cells out of the total tracked cells (example in Supplementary Fig. 1d, e). A total of 4 biologically independent experiments, 120 cells/group, were analyzed by investigators blinded to experimental conditions/groups.

PathScan intracellular signaling array and Western blotting
Intracellular signaling arrays (PathScan, Cell Signaling Technology, 7744) were analyzed from previously published data20. The data shown are from hNSC treated with C1q at 0.1 nM, 1.0 nM, or 200 nM concentrations for 60 min, normalized to internal positive controls and non-treated hNSC controls.

Western blot analyses were performed using protein lysates collected from non-treated, C1q or temperature-inactivated C1q-treated hNSC on 6-well plates. Band densities were quantified using ImageJ (NIH) and normalized to loading controls β-actin, α-tubulin, or total p53 (phospho-p53Ser15) and non-treated controls. Each biologically independent experiment consists of one well. Primary antibodies and dilutions used were as follows: Tubulin β3 (1:500, Biolegend, TUBB3, 801202), Glial Fibrillary Acidic Protein (1:5000, Dako, Z 0334AB_10013382), Glial Fibrillary Acidic Protein δ (1:500, Millipore, AB9598), S100β (1:200, Abcam, Ab52642), vimentin (1:1000, Abcam, Ab24525), nestin (1:1000, Abcam, Ab11306), SOX2 (1:200, R&D systems, AF2018), Phospho-p53 (Ser15) (1:300, Cell Signaling, 16G8, 9286), p53 (1:500, Cell Signaling, 9282s), GC1qR (p32) (1:100, Abcam, Ab24733), MTCO1 COXIV (1:1000, Invitrogen, 459600), BAI1 (1:1000, R&D systems, AF4969), BAI1 (1:1000, Novus Biologicals, NB110-81586), BAI1 polyclonal antibody (1:1000, Invitrogen, PA5-102069), β-actin (1:2500, Sigma-Aldrich, A1978), or α-tubulin (1:7000, Abcam, DM1A, Ab7291). For pulldown analysis, we used antibodies against BAI1 (1:1000, R&D systems, AF4969), C1q (1:500, Abcam, Ab71940), and p32 (1:2000, Abcam, Ab24733). Secondary antibodies used were horseradish peroxidase (HRP)-conjugated secondary antibodies at 1:5000 dilution (Fisher Scientific, NA931, NA934, or NA935) or (Thermo Fisher, 31402, A16041, or A16011). For quantitative comparisons, experimental control and sample lanes, as well as loading controls for each biologically independent experiment, were analyzed from the same membranes. Samples with technical errors were excluded from the study. Normalized phospho-p53Ser15 and p32 bands/independent experiment were tested for outliers using Grubb’s outlier test (Alpha 0.2) in Prism. Exclusions, outliers, and unprocessed scans of the most important blots with loading controls are provided in the Source Data.

RNAseq and single-cell RNAseq
hNSC were plated at 500,000 cells/well as a monolayer in 6-well plates and cultured with or without 200 nM C1q in differentiation medium (DM) for 48 h. Each biologically independent experiment consists of one well. RNA was extracted and quantified for quality (Supplementary Methods). Library construction and sequencing were carried out at the UCI GHTF using a TruSeq RNA Library Prep Kit and Illumina HiSeq 4000 (Illumina), generating ~30 million paired-end reads. Before read mapping, each sample was quality-checked with FastQC100. Ribosomal contamination was removed using the SortMeRNA101, and low-quality bases (Q<30) were filtered out with Trimmomatic tool102. RNAseq reads were mapped to the human genome GRCh38 version 105 using the STAR aligner, uniquely mapped reads were summarized using featureCounts103 and differential gene expression analysis was conducted on TMM normalized counts using EdgeR104. Significant genes were identified with an FDR-adjusted p≤0.05. Gene Set Enrichment Analysis (GSEA version 4.3.2)105 was performed using the hallmark gene set collection from Molecular Signature Database (MSigDB)106. Pathway enrichment analysis of quiescence, activation, and neural differentiation-associated transcripts was analyzed using GSEA for each manually curated gene set. Normalized enrichment scores (NES) were visualized using the ggradar R package. Gene sets between the groups were considered statistically significant at a multiple comparison p-value of 0.05 and visualized using heatmaps. All analyses were performed by investigators blinded to experimental conditions/groups. Local C1q expression by different spinal cord cell types in intact mice vs. 1, 3, or 7 d post-SCI was analyzed using previously published scRNAseq data70.

CRISPR Cas9 hNSC BAI1 genetic deletion
Tissue-derived multipotent hNSC are a heterogeneous stem cell population that in long-term culture with an increasing number of passages exhibit an increased risk for undesirable profile changes to a gliogenic phenotype. Therefore, isogenic clone selection carries a heightened risk of inadvertent deletion of cell subpopulations or types. To address this, we developed a two-stage genetic modification strategy using CRISPR cas9 and FACS to create BAI1 WT and KO hNSC. Based on our initial analysis, 80% of unmodified hNSC were BAI1+, and 20% were BAI1-.

Using a custom BAI1 donor template (Supplementary Fig. 2a) and a BAI1 CRISPR plasmid (Applied Stem Cells, cat no. P0049-P001), we introduced premature stop codons into first open reading frame of BAI1 within exon 2 - along with a floxed CMV-mScarlet-P2A-PuroR cassette leading to truncated BAI1 at amino acid 52, reducing the size of BAI1 by 96.7% to effectively create precise genetic KO in hNSC (Supplementary Fig. 2b). After positive selection of mScarlet-expressing KO hNSC using FACS, CRE recombinase was added to BAI1 KO hNSC to remove the selection cassette (Supplementary Fig. 2b), causing the BAI1 KO hNSC to become mScarlet-negative. The cas9-expressing-BAI1 Guide RNA plasmid (gRNA sequence in Supplementary Fig. 2c) was custom-generated and validated in human HEK293 cells by Applied Stem Cells (cat no. P0049-P001). The BAI1 donor plasmid (Supplementary Fig. 2a) was constructed in-house (Supplementary Methods). Homozygous insertion of the frame-shift premature stop codons in BAI1 KO hNSC was verified via PCR of genomic DNA and Sanger sequencing (Supplementary Fig. 2d, e). Stable loss of total BAI1 protein (174 kDa) expression was confirmed under non-reducing conditions by Western blotting (Fig. 3b), and functional level, by performing PLA specific for the protein-protein interaction of BAI1 with its ligand, C1q, in both BAI1 KO and WT cells (Fig. 3c, d). Two different BAI1 KO lines were generated with the same method. No substantial differences between the cell lines generated were detected regarding their CD133+ content, multipotency, migration, or proliferative capacity. BAI1 KO hNSC characterization is detailed in the Supplementary Methods.

Proximity ligation assays
hNSC were plated on PLO-laminin or biolaminin (BioLamina, CT521-0501) coated 8-well glass chamber slides and treated with 100 nM or 200 nM C1q or equal volume of GM (non-treated control) for 30 min or 1 h as previously described in ref. 20. All biologically independent experiments were performed with technical duplicates. PFA 4%-fixed and wheat germ agglutinin (WGA)-conjugate 488 (Invitrogen) stained cells were permeabilized and blocked with 0.01% Triton X (Sigma Aldrich) and 2% Donkey Serum (Jackson Immunoresearch). PLA was performed using Duolink® In Situ PLA® Probes (Supplementary Methods) following the manufacturer instructions. PLA signal, presented as total punctae per cell with a positive signal, was quantified using Imaris from z-stacks of optical slices captured at 0.3 μm intervals using a 60x objective. Primary antibodies used were anti-C1q (1:100, Abcam, Ab71940), anti-BAI1 (1:500, Abcam, Ab135907), anti-MDM2 (1:100, Santacruz biotechnology, SMP14, Sc-965), recombinant anti-Met (c-Met) (1:500, Abcam, Ab51067), or gC1qR rabbit (1:500, Abcam, ab270033). All analyses were performed by investigators blinded to experimental conditions/groups.

Immunocytochemistry and flow cytometry
hNSC monolayers fixed with 4% PFA were permeabilized and immunostained with primary antibodies against cleaved caspase-3 (1:100, Cell Signaling, Asp175, D3E9, Cat. No. 9602s), phospho-p53 (Ser15) (1:400, Cell Signaling 16G8, 9286s), recombinant anti-GC1qR (p32) (1:200, Abcam, Ab24733), or TOM20 (1:200, Cell Signaling, D8T4N, 42406S). Secondary antibodies conjugated with 555, 488, or 647 fluorochromes were used at 1:2000, 1:1000, or 1:500 dilution, respectively (Invitrogen, A131570, A312572, A21202, A21206, A31571, A31573, or A21447), and Hoechst 33342 (1 µg/mL; Invitrogen, H3570) was used as a nuclear counterstain. All biologically independent experiments were performed with technical duplicates. Images captured using a LSM900 (Zeiss) or BZ-X (Keyence) microscope with a 20x objective were analyzed with Imaris by investigators blinded to experimental conditions/groups. For phospho-p53Ser15 quantification, the cells with only nuclear label were considered positive. Samples with technical issues during the staining, such as drying out of the wells or wells with insufficient amount of antibody, were excluded from the analysis. Exclusions and outliers are listed in the Source Data.

For imaging flow cytometry, hNSC were plated on PLO-laminin-coated flasks and treated with 200 nM C1q or an equal volume of GM (non-treated control) for 5 min, 15 min, 30 min, 1 h, 2 h, or 24 h. For testing C1q colocalization in endosomes or Golgi, hNSC were transfected with either CellLight™ Early Endosomes-RFP (Invitrogen, C10587), or Golgi-RFP (Invitrogen, C10593). The next day, cells were washed and treated with 200 nM C1q for 24 h. Details for matched positive and mismatched negative ligand-receptor controls (Fig. 4j, k and Supplementary Fig. 4b–j) are in the Supplementary Methods. After treatment, cells were washed and detached using Trypsin/EDTA or non-enzymatic cell dissociation solution (Sigma-Aldrich, C5914). After treatment, the cells were aliquoted at a density of 20,000 cells/μL per tube and fixed using 2% PFA for 15 min on ice, followed by wash with 1% fish gelatin in 0.1 M TBS, blocking, and 1 min permeabilization using DPBS supplemented with 0.1% or 0.02% Triton X-100, 5% goat or donkey serum. Primary antibodies diluted in 0.1 M TBS wash buffer with 1% fish gelatin and 5% serum were incubated with cells overnight at +4°C. Primary antibodies used were anti-C1q (1:50, Abcam, Ab71940), anti-human BAI1 (1:50, R&D systems, AF4969), anti-BAI1 (1:50, Lifespan Biosciences, LS-C120632), anti-human complement C3a (1:50, Millipore/Chemicon, CBL191), anti-C3aR (1:50, Abcam, Ab103629), anti-EGFR (Abcam, EP38y, ab52894), recombinant anti-GC1qR (1:50, Abcam, Ab24733). Secondary antibodies used (1:500) were conjugated with 555, 488, or 647 fluorochromes (Invitrogen, A131570, A312572, A21202, A21206, A21141, A31571, A31573, A21240, or A21447). The BAI1 antibodies used for Imagestream, Western blotting, or PLA, all recognize the full-length BAI1 when evaluated using BAI1 overexpression lysate and empty vector negative HEK293T control lysate (Novus Biologicals; Supplementary Fig. 3a). Single cells were imaged using Amnis Imagestream x Mark II Imaging Flow Cytometer and analyzed using IDEAS version 6.2 (Cytek Biosciences)51,107 and component masking (Fig. 4e) according to the manufacturer instructions. Analysis details are available in the Supplementary Methods. Gating (Supplementary Fig. 3b, c) were set using unstained negative control cells, secondary antibody only controls, single-labeled cells, and antibody compensation beads. For protein internalization, score ≥0.5 was considered internalized. Mean bright detail similarity R3 score ≥1.5 was considered colocalized. Spot count feature (Supplementary Fig. 4o) was used for measuring the quantity of internalized protein per cell. Time-course data was collected from one to four biologically independent experiments with one sample per each. Each biologically independent experiment consists of one flask.

Immunogold electron microscopy
For electron microscopy, hNSC were cultured on 8-well chamber slides with or without C1q for 24 h. After washing with 0.1 M phosphate buffer, cells were fixed with a mixture of 0.25% glutaraldehyde (Ted Pella, Inc., #18420) in 2% PFA (Polysciences, #00380) in 0.1 M PB with 50 mM sucrose and 0.4 mM CaCl2 on ice followed by washes, blocking, and quenching with 1% NaBH4 in 0.01 M PBS. Cells were permeabilized with 0.2% Triton-X 100 and incubated with anti-C1q antibody (1:50; Abcam, Ab71940) overnight at 4°C. The next day, nonspecific binding sites were blocked with wash buffer and incubated with 1.4 nm Nanogold-antimouse Fab’ fragment (1:50, Nanoprobes Inc., #2022) at room temperature for 2 h followed by fixation with 1% glutaraldehyde. Immunogold deposits were enhanced by incubation with ionic silver in citrate buffer (HQ Silver Enhancer, Nanoprobes Inc., #2012) followed by washes, post-fixing with 2% OsO4, dehydration, and embedding in Epon. Sections cut at 80 nm thickness were counterstained with 4% uranyl acetate and lead citrate and examined with a JEOL 100CX transmission electron microscope (JEOL USA). Negatives were scanned using an AGFA Duoscan T1200, and images were processed using Photoshop (Adobe Systems). Data represents two biologically independent experiments with technical duplicates. All analyses were performed by investigators blinded to experimental conditions/groups.

C1q biotinylation, cross-linking, co-immunoprecipitation, and LC-MS/MS
C1q was biotinylated using cell membrane-permeable EZ-Link NHS-SS-Biotin (Thermo Scientific, PI21441). Biotinylated C1q (200 nM) or growth medium (control) was applied to cells for 15 min or 30 min at RT. Analysis was performed as four biologically independent experiments with one sample well per each. Intracellular crosslinking was performed with DSP (Thermo Scientific, PI22586), followed by lysis in Pierce IP buffer (Thermo Scientific, PI87787) and pull-down on Streptavidin Magnetic Beads (Thermo Scientific, PI188816). Step-by-step protocols describing the procedures used in this study for have been deposited on protocols.io and can be accessed as follows: C1q biotinylation, cross-linking, and co-immunoprecipitation/pull-down dx.doi.org/10.17504/protocols.io.14egnrydpl5d/v1, denaturing on-bead-digestion dx.doi.org/10.17504/protocols.io.bp2l6zp91gqe/v1, and desalting dx.doi.org/10.17504/protocols.io.rm7vz92bxgx1/v1. Peptide digests were analyzed using an UltiMate 3000 RSLC system coupled in-line to an Orbitrap Fusion Lumos mass spectrometer. Separation was performed on a 50 cm Acclaim® PepMap RSLC column with a 4% to 22% gradient over 70 min at 300 nL/min. Mass spectra were acquired in a cycle of full Fourier transform scans (375–1500 m/z, resolution 120,000) followed by data-dependent MS/MS scans with HCD at 30% NCE. Ions selected for MS/MS were dynamically excluded for 30 s. Protein identification and label-free quantitation were performed using MaxQuant version 1.6.0.16109. Further details available in the Supplementary Methods. Peptide spectra match and protein FDR were both set to 0.01. Data analysis utilized Differential Enrichment Pipeline (Bioconductor, DEP version 1.22)110. Samples treated with biotinylated C1q per each biologically independent experiment were pooled together and protein-wise linear models combined with empirical Bayesian statistics were used to identify differentially expressed proteins between bait versus non-treated control (FDR-adjusted p-values≤0.05). Subcellular localization and enrichment were assessed using SubCellViz111 and hypergeometric tests, identifying significantly enriched compartments associated with receptor-mediated endocytic trafficking by identified prey proteins (Supplementary Table 2). All analyses were performed by investigators blinded to experimental conditions/groups.

Protein enrichment of specific cellular compartments and pull-down protein interaction analysis
To evaluate C1q effect on total p32 protein or p32 protein availability on hNSC surface, the cell surface proteins were enriched using non-cell membrane permeable EZ-Link™ Sulfo-NHS-SS-Biotin (Thermo Scientific, 21331) and pull-down was performed using (Thermo Scientific, Streptavidin Agarose Resin, 20347). Extraction of cytoplasmic, membrane/structural, nuclear, and insoluble proteins from hNSC was performed using a sequential detergent- and salt-based subcellular fractionation. Analyses were performed as a single biologically independent experiment to validate the Imagestream and immunocytochemistry data (Supplementary fig. 4p, q). Step-by-step protocols describing the procedures used in this study for have been deposited on protocols.io and can be accessed as follows: Enrichment of surface proteins using biotin dx.doi.org/10.17504/protocols.io.rm7vz9qz4gx1/v1, and subcellular fractionation dx.doi.org/10.17504/protocols.io.q26g7n9o8lwz/v1.

Direct interactions among C1q, BAI1, and p32 were tested using His-tagged recombinant BAI1 or p32 (Sino Biological) as bait and purified or untagged C1q/p32 (MyBioSource, LSBio) as prey. Pull-downs were performed with Dynabeads His-Tag Isolation (Invitrogen, 10103D) in two biologically independent experiments. A step-by-step protocol describing in situ pull-down assay to test direct interaction between C1q, BAI1 and p32 has been deposited on protocols.io and can be accessed through: dx.doi.org/10.17504/protocols.io.kxygx4qjzl8j/v1. The original blots are available in the Source Data.

Two-photon fluorescence lifetime microscopy and phasor data
NADH/NAD+ redox ratio indicating glycolytic and OXPHOS states in hNSC was measured using a label-free, phasor approach with two-photon FLIM as previously described64–66. hNSC were cultured overnight on PLO-laminin-coated 8-well coverslip slides and then treated with or without C1q for time course analysis. FLIM images were captured using a two-photon microscope with a Ti:Sapphire laser (Spectra-Physics Mai Tai) and a Zeiss Axiovert S100TV 40x objective at 740 nm. A dichroic filter (700 DCSPXR, Chroma Technologies) a hybrid detector (HPM-100, Hamamatsu) was used for separating and detecting the signal. Fluorescein lifetime was used for calibrating the detection, and the data was processed using SimFCS software (UCI Laboratory for Fluorescence Dynamics). Data from 5-9 cells over 3 biologically independent experiments were analyzed and phasor plot values calculated for each cell. All analyses were performed by investigators blinded to experimental conditions/groups.

Mitochondria segmentation and morphology
hNSC were cultured on PLO-laminin-coated 8-well slides in GM overnight. The next day, cells were treated with 200 nM C1q or equivalent amount of GM (non-treated control) for 24 h. Cells were washed and stained with 100 nM MitoTracker™ Red CMXRos (Invitrogen, M7512) for 30 min. Live cell imaging was performed in pre-warmed GM using a Zeiss LSM900 Airyscan with a 60x objective and z-stacks of 0.5 µm optical sections. Mitochondria were segmented and analyzed with CellProfiler version 4.2.1 (CellProfiler) and MitoMo (https://www.vislab.ucr.edu/SOFTWARE/software.php) followed by Matlab (MathWorks) analysis as previously described108. All biologically independent experiments were performed with technical duplicates. Analyses were performed by investigators blinded to experimental conditions/groups. Values of each independent experiment were tested for outliers using Grubb’s outlier test (Alpha 0.2) in Prism; outliers are listed in the Source Data.

Seahorse mito stress test
hNSC were seeded at 40,000 cells/well on PLO-laminin-coated XF24 microplates (Agilent Technologies). The next day, cells were treated with 200 nM C1q or equivalent amount of GM for 48 h. Metabolic status was assessed using the Seahorse XF24 Cell Mito Stress Test Kit and Seahorse XF24 extracellular flux analyzer (Agilent Technologies) according to the manufacturer instructions. More details available in the Supplementary Methods. Data were analyzed Wave version 2.6.1 (Agilent Technologies) and normalized to total protein of respective sample wells measured using Qubit (Thermo Fisher Scientific, Q33211). All biologically independent experiments were performed with at least technical triplicates. Wells with technical errors were excluded from the analysis. Basal OCR and ECAR are means of three basal reads per group per each independent experiment. Normalized values for each experiment were tested for outliers using Grubb’s outlier test (Alpha 0.05) in Prism; exclusions and outliers are listed in the Source Data.

Spinal cord injury, stem cell transplantation, and histological analysis
10-week-old Rag-1-immunodeficient female mice (Jackson Laboratories, B6; 129S7-Rag1tm1Mom/J, #002096) (n=14) received a moderate 60 kD contusion injury at vertebral T9 under anesthesia as previously described in refs. 109–112. Immediately after injury, mice received four 250 nL injections (total volume 1 μL; 75,000 cells) of either BAI1 WT or BAI1 KO hNSC into spinal cord intact parenchyma adjacent to lesion epicenter as previously described in refs. 109–112. All mice received standard post-operative care, including Ringer’s lactate solution (MWI Animal Health), Bubrenorphine (MWI Animal Health), Baytril (Patterson Veterinary) SQ, and twice a day bladder care. To label proliferating cells, mice were given 50 mg/kg BrdU i.p. twice a day until the end of the study 2 week post-injury/transplant (wpt).

For histological analysis, mice were anesthetized and perfused with 4% PFA at 2 wpt, and T6–T12 spinal cord segment was dissected, post-fixed in 20% sucrose/4% PFA, and frozen in isopentane at −65°C. Transverse sections were cut at 30 μm using a CryoJane tape transfer system (Leica Microsystems). Sections in the 1/12 sampling sequence were processed for antigen retrieval using a citrate buffer (PickCell Laboratories, Retriever 2100). Antibodies and dilutions used were human-specific STEM121 (1:2000, Takara, AB281314) and BrdU (1:200, Abcam, Ab1893) antibodies. Unbiased stereological analysis was conducted on an Olympus IX51 microscope with a 60x objective using systematic random sampling, an optical fractionator probe, and Stereo Investigator version 9 (MicroBrightField) as previously described in refs. 111, 112. The sampling was designed to meet stereological validation principles. Animal care and analysis were performed by investigators blinded to experimental conditions/groups.

Statistics and reproducibility
Statistical analysis methodology including corresponding details can be found both within the Figure Legends and in the Source Data. All data are presented as mean ± standard mean of error (s.e.m). Number of biologically independent replicates or measurements referring to numbers each experiment was repeated independently with similar results are listed in the Figure legends and in the Source Data. Before conducting statistical analyses, the data were assessed for normality and homogeneity of variances, and appropriate parametric or non-parametric tests were applied as outlined in the Source Data or Supplementary Source Data.

Ethics statement
All human stem cell experiments were approved by the Human Stem Cell Research Oversight committee (#3682) and Institutional Biosafety committee (BUA-R120) at UCI. Informed consent was obtained from all fetal tissue donors. Mouse procedures and mNSC derivation followed UCI Institutional Animal Care and Use Committee (IACUC; AUP-23-023) and NIH animal care guidelines.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript reports effects of C1q on human neural stem cells (hNSCs) mediated by the receptor BAI1. The authors describe effects of C1q on hNSC proliferation and other aspects of the cells’ physiology. In parallel studies, the authors provide insight into the signaling pathways that are engaged when C1q binds BAI1, specifically i) regulation of mdm2 to control p53 and ii) endocytosis of C1q to modulate p32. These findings are novel, although the connections between the signaling pathways elucidated and the effects of C1q on hNSC proliferation are more hypothetical than directly shown. Specific comments are as follows:\n1. The central physiological effect reported by the authors is a striking effect of C1q on hNSC proliferation (Fig. 1a). The authors also provide evidence that this effect is mediated by C1q engagement of BAI1 (Fig. 6). Separately, the authors provide evidence that C1q can regulate p53 levels in hNSCs (Fig. 3) and additionally regulate p32 and aerobic glycolysis after gaining entry into hNSCs via BAI1 (Fig. 4-5). However, it is not clear from the data presented whether these mechanisms actually account for the striking effect of C1q on hNSC proliferation shown in Figure 1. To put it another way: Figs. 1 & 6 show that C1q regulates hNSC proliferation in a BAI1-dependent manner, and the data from Fig. 3-5 provide insight into various signaling pathways that are activated when C1q engages BAI1 in hNSCs. What’s not shown, though, is whether these signaling pathways are directly responsible for the effect of C1q on hNSC proliferation. Given that BAI1 is known to couple to other signaling pathways (Rac, Rho, etc) that are capable of affecting proliferation, the authors need to support their proposed model by more definitively demonstrating that the mechanisms they describe (regulation of p53 and p32) are in fact responsible for directly mediating the ability of C1q to regulate hNSC proliferation.\n2. In creating the BAI1 KO cell line, the authors show the proper DNA fragment sizes (Extended Data, Figure 2). However, in the Western blot data shown in Fig. 3b, there are still BAI1-positive bands in the BAI1 KO lanes, which means that BAI1 may not actually be deleted at the protein level. The authors should address this point. If the authors believe these bands in the KO lanes to be non-specific, they should state what evidence leads them to this conclusion. This is an important issue to resolve, given that many of the authors’ conclusions depend on BAI1 being fully deleted from the KO cell line.\n3. For the experiments with the BAI1 neutralizing antibody (nAb) shown in Fig. 6, it is unclear which BAI1 antibody was used by the authors for these experiments. Also, how was this antibody characterized? How do the authors know that it is truly a neutralizing antibody? Obviously, it is possible for antibodies to bind to a receptor but not neutralize ligand binding, and thus it is important for the authors to provide more information about the characterization of this antibody. The authors provide a citation for a previous characterization of the C1q nAb that was used in these studies, but no comparable citation is provided for the BAI1 nAb.", "reviewer_2": "Review of the manuscript entitled “C1q drives neural stem cell quiescence by regulating cell cycle and metabolism through BAI1”.\nIn this manuscript, the authors describe a role for the complement-derived pattern recognition molecule C1q in neural stem cell (NSC) quiescence. The team had previously identified several new receptors for C1q, including brain angiogenesis inhibitor (BAI1). Here, they follow up this functional interaction and demonstrate C1q binding to BAI1 on NSCs induces, at minimum, two events: firstly, BAI1-mediated sequestering of MDM2 which, in turn, increases p53 phosphorylation and blocks cell cycle progression an secondly, BAI1-depenent internalization of C1q which enables interaction of C1q with the mitochondrial C1q receptor p32. The latter than induces a shift from OXPHOS to aerobic glycolysis which also supports quiescence. As a general model, the authors argue that age- or inflammation/trauma induced increases in local C1q production may sustain NSC quiescence and inhibit activation when desired (during tissue repair, etc.).\nThe team makes its case using a set of elegant and detailed in vitro experiments and argues that this pathway is important in vivo by showing that the engraftment of BAI-KO human NSC into the brains of mice exposed to an acute CNS injury model (which is associated with local increases in C1q) rescues the decline in hNSC proliferation when compared to WT hNSC.\nOverall, the study is interesting and timely and its strength lays in the fact that it further explored a new C1q-receptor interaction. Given the emerging central role for C1q across human disease and specifically in CNS pathology settings, C1q is becoming a valuable therapeutic target. The large majority in the field, however, focusses on the ‘canonical’ C1q-repetior interactions (gC1qR and cC1qR). C1q biology is highly complex, and it is therefore paramount to better understand its biology for successful future targeted interventions. This leads to the second strength of the work: the authors argue that the effects of C1q in each tissue will depend on the C1q receptor ‘availability’ which then also dictates the specific outcome of C1q activity. This aligns well with the emerging realization that local complement impact is highly cell and context specific.\nThere are several areas where the work can (and needs to be improved):\n1. A major issue is that the authors have not satisfactorily dissected the effects on proliferation, self-renewal, cell death and survival. Particularly Extended data Figure 3B and C is difficult to reconcile as the control phenotype seems to be driven by a single cell that divides extensively.\n2. Although the authors likely want to make it easier ‘on the eye’ by showing the control data as a 100% threshold line, raw data are preferred to see the spread here.\n3. In general, all experiment dealing with phosphorylated proteins (p53 etc.) need to show total protein as comparator).\n4. The mitochondrial staining for C1q/p32 etc. are not convincing and should be repeated in high resolution (like Figure 5I, which is very nice).\n5. The authors argue that BAL1 induces C1q internalization which then can bind to p32 as it may be released. However, there should be a high-resolution triple staining for C1q/BAL1/p32 (which should be negative) to support this model.\n6. Figure 1E: something is not right here with the last 2 conditions: why does the isotype control IgG antibody further amplify the reducing effect of C1q on NSC proliferation and also has such a strong inhibitory effect without C1q addition?\n7. Does ERK inhibition have similar effects on NSC proliferation?\n8. Figure 4K: the negative C3aR/C1q control looks positive (?) or has a very high background which makes it difficult to interpret the other data – thoughts?\n9. Figure 7: the analysis of C1q is very nice – why did the authors not perform the same analysis for BAI1? It would be nice to see the expression profile of this C1q receptor in relation to its ligand. It would have also added some suggestions to whether the interaction by be autocrine or paracrine.\n10. The in vivo experiment in Figure 7 solidifies the role of BAL1 in hNSC proliferation control. It does not conclusively allow to associate this with the C1q activity though.\nCould the authors approach this somehow? For example, could there be a bulk RNA-seq analysis of in vitro WT and BAL1 KO hNST +/- C1q (as in Figure 6) to generate C1q-BAL1 axis (normal/perturbation) signatures and then utilize these signatures to assess the bulk RNA-seq signatures from re-isolated SC121-Brdu+ cells WT/KO cells in the lesions (Figure 7D)?\n11. The manuscript has many errors when it comes to labeling and cross-referencing figures and data. For example, the table is references erroneously as Extended Data 2, there is no reference to Extended Data 3 in the text and Extended Data 8 (mentioned in the text) are entirely missing, Extended Data 7 legend seems not to fit (ROS), etc.. Overall, the structure of text, figures, and Extended data can be improved – it was at times difficult to navigate text and figures.", "reviewer_3": "In this work the authors focus on the complement factor C1q and its effects on proliferation and quiescence of human neural stem cells (hNSCs). First, they demonstrate that C1q treatment leads to these cells proliferating significantly less with the absence of any changes in apoptosis, suggesting an induction of quiescence. Next, they undertake a variety of studies to assess the underlying mechanism of this effect. The first of these mechanisms is shown through changes in p53 phosphorylation and localization to the nucleus which seems to occur through MDM2 initiated by binding to BAI1. They then assess the internalization of C1q and suggest that this occurs via endocytosis. The authors also detail a novel role for C1q in mitochondrial changes in hNSCs acting through p32 and BAI1. Finally, the authors show that loss of BAI1 rescues the C1q impairments in proliferation and that transplantation of BAI1 KO cells are more highly proliferative in a spinal cord injury model. These data are exciting and expand our understanding of the role of immune molecules in the control of proliferation and quiescence of neural stem cell populations. While I am enthusiastic about these studies, I do have a number of criticisms that the authors should consider.\n-In Figure 1E, it is shown that IgG nab both alone or in the presence of C1q leads to a ~5-fold larger reduction in BrdU positive cells than the when C1q is used alone. How can this be as this is a control antibody? Moreover, in this same figure it’s shown that after priming for 4-8 weeks this actually leads to greater proliferation (ie. BrdU+ cells) than control. Can the authors explain why this might be? Are the spheres just much larger but there are many fold less of them?\n-In a number of cases, western blots are shown with bands that come from a number of different blots including when trying to demonstrate quantitative differences (for example Figure 1K). In such cases, these data need to come from one blot.\n-In multiple figures (for example, Figure 2c and Figure 3d) no control images are shown and only treatment examples are used. This makes interpretation of the data difficult. In the case of Figure 2c, there is an apparent 4-fold increase in the presence of C1q which should be striking in images comparing Control to 200 nM C1q.\n-While the BAI1 KO and C1q dependence in this work is convincing the lack of a direct demonstration of C1q and BAI1 interaction is a significant weakness. Considering that BAI1 recombinant protein is commercially available it should be straightforward to demonstrate a direct interaction using a technique such as biolayer interferometry or surface plasmon resonance or a more traditional pull-down assay. It remains possible that the interaction between BAI1 and C1q is indirect.\n-The model with which p32 is involved with C1q and BAI1 doesn’t seem fully plausible. The author’s own data suggests that p32 is in the mitochondrial matrix while C1q in large part enters the cell via endosomes. How can these two proteins specifically interact when they are in different cellular compartments? This would require demonstrating that both C1q are in the same place at the same time in hNSCs at high resolution. Moreover, if the authors propose, as in Figure 5e, that C1q enters endosomes and then is released from endosomes this would need to be demonstrated experimentally.\nMinor:\n-Page 2 Line 1 BCB not defined. Perhaps BSB typo?\n-In a number of cases “Blood-derived C1q concentrations” is used. This is confusing as the data shown here actually has nothing to do with “Blood-derived C1q.” Consider modifying this terminology for clarity.\n-In Figure 3B, this is the only blot shown as white bands on black background when all of the rest of the blots are shown inverted. This is confusing and can lead to confusion over whether these data are western or an agarose gel.\n-In Figure 4i, pull down based proteomics data is introduced but these data are hardly described or mentioned at all and only shown as a cartoon. If these data are significant enough to be shown in the main figures they should be described and interpreted in a more fulsome manner.", "reviewer_4": "\"I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\""}, "author_response": "We appreciate the valuable feedback on our manuscript, and the time and effort\ninvested in providing detailed comments and suggestions. We have carefully\nconsidered each comment and made revisions accordingly. Additionally, as requested,\nall of the figures have been formatted to match Nature Communications requirements,\nand the source data files with raw data have been included in this resubmission. Below,\nwe address each of the points raised.\nReviewer #1 (Remarks to the Author):\nThis manuscript reports effects of C1q on human neural stem cells (hNSCs) mediated\nby the receptor BAI1. The authors describe effects of C1q on hNSC proliferation and\nother aspects of the cells’ physiology. In parallel studies, the authors provide insight into\nthe signaling pathways that are engaged when C1q binds BAI1, specifically i) regulation\nof mdm2 to control p53 and ii) endocytosis of C1q to modulate p32. These findings are\nnovel, although the connections between the signaling pathways elucidated and the\neffects of C1q on hNSC proliferation are more hypothetical than directly shown. Specific\ncomments are as follows:\n1. The central physiological effect reported by the authors is a striking effect of C1q on\nhNSC proliferation (Fig. 1a). The authors also provide evidence that this effect is\nmediated by C1q engagement of BAI1 (Fig. 6). Separately, the authors provide\nevidence that C1q can regulate p53 levels in hNSCs (Fig. 3) and additionally\nregulate p32 and aerobic glycolysis after gaining entry into hNSCs via BAI1 (Fig.\n4-5). However, it is not clear from the data presented whether these mechanisms\nactually account for the striking effect of C1q on hNSC proliferation shown in Figure\n1. To put it another way: Figs. 1 & 6 show that C1q regulates hNSC proliferation in a\nBAI1-dependent manner, and the data from Fig. 3-5 provide insight into various\nsignaling pathways that are activated when C1q engages BAI1 in hNSCs. What’s\nnot shown, though, is whether these signaling pathways are directly responsible for\nthe effect of C1q on hNSC proliferation. Given that BAI1 is known to couple to other\nsignaling pathways (Rac, Rho, etc) that are capable of affecting proliferation, the\nauthors need to support their proposed model by more definitively demonstrating\nthat the mechanisms they describe (regulation of p53 and p32) are in fact\nresponsible for directly mediating the ability of C1q to regulate hNSC proliferation.\nWe have previously published that multiple intracellular signaling pathways contribute to\nto C1q-driven proliferation in hNSC (PMID: 32894219). However, in our phosphoarray\ndata, the only unique sustained signaling pathway in C1q [200nM]-treated hNSC was an\nincrease in phosphorylation of p53 serine 15 (p53Ser15) (Fig.2a; PMID: 32894219); due\nto the previously published link between p53 and BAI1 in tumor cell proliferation\n(PMID: 29894688), we therefore focused on testing the effect and mechanisms of C1q-\nBAI1 interaction in hNSC proliferation. We appreciate Reviewer’s interest in extending\nthe identification of a novel role for C1q-BAI1 interaction in hNSC proliferation to include\n1\nestablishment of a direct role for p53 and p32 signaling in this mechanism, however,\nattempts to so so have proven impossible.\nMost critically, in our paradigm, disruption of either of p32 or p53 signaling leads to rapid\ncell death in hNSC. This is consistent with reports that p32 KO in mammalian systems is\nembryonic lethal before the peak of neurogenesis (PMID: 22904065); thus, analysis of\nthe direct roles of p32 would require use of non-mammalian model systems\n(PMID: 36995025). Towards that goal, we attempted to establish a neural cell paradigm\nin drosophila, in which p32 knockout has been reported, but were unable to produce\nviable cells. We also attempted to manipulate p53 signaling using the selective chemical\ninhibitor UC2288 (Sigma-Aldrich, 532813), which targets the downstream effector p21\n(PMID: 23298903). However, consistent with the literature (PMID: 27156098), p53\npathway inhibition with UC2288 during C1q-treatment of hNSC induced apoptosis\n(Response letter_Fig.1). The negative impact of p32 and p53 signaling inhibition on\ndividing cells is not surprising, due to the energy requirements of these cells and role of\np32 in mitochondrial function, as well as the expected effect of disrupting the cell cycle\nin a dividing cell. As a result, with the current tools available, there does not appear to\nbe a viable approach by which to reasonably be able to address this review comment.\nFinally, we would like highlight that, as we discuss in the manuscript (page 8, lines\n38-42 and page 9, lines 1-3), we do not propose that hNSC proliferation is uniquely\nmodulated by C1q-BAI1 interaction, and predict that other putative C1q receptors may\nplay roles in regulating this crucial biological process. Hence, while dissecting\npotentially converging signaling pathways may ultimately be required, we suggest that\nthe fundamental novelty of the current findings remains an important contribution to the\nliterature.\nResponse letter_Fig.1. Selective chemical inhibitor UC2288 targeting p53\ndownstream effector p21 increases hNSC death (CC3+ cells) relative to\ndifferentiation medium (DM) + DMSO controls (dash line) in C1q 200nM-\ntreated hNSC.\n2\n2. In creating the BAI1 KO cell line, the authors show the proper DNA fragment sizes\n(Extended Data, Figure 2). However, in the Western blot data shown in Fig. 3b, there\nare still BAI1-positive bands in the BAI1 KO lanes, which means that BAI1 may not\nactually be deleted at the protein level. The authors should address this point. If the\nauthors believe these bands in the KO lanes to be non-specific, they should state what\nevidence leads them to this conclusion. This is an important issue to resolve, given that\nmany of the authors’ conclusions depend on BAI1 being fully deleted from the KO cell\nline.\nThank you for your detailed comments regarding our hNSC BAI1 knockout (KO) line –\nwe sincerely appreciate the opportunity to address these concerns. To clarify, at the end\nof our two-step genome editing strategy we created a precise genetic KO of the BAI1\ngene in hNSCs by inserting an in-frame loss-of-function mutational scar (consisting of\nstop codons) into the start of the BAI1 open reading frame at amino acid 52 of 1584\ntotal (96.7% size reduction). In this manuscript we present three lines of orthogonal\nevidence that demonstrate the successful silencing of BAI1 in our KO line as discussed\nbelow.\nFirst, at the genetic-level, if the editing was successful, it is expected that the mutational\nscar at the BAI1 loci would increase the size of amplicons around the editing site by\n44BP in KO cells only in comparison to the unedited alleles of wild-type (WT) cells.\nIn Supplementary Fig. 2, we clearly demonstrate that we generated precise genetic BAI1\nKO cells via sanger sequencing – providing the first line of evidence.\nSecond we believe we may have created confusion by including both reducing and non-\nreducing gel images in the main figures, and have refined the figure accordingly. All\nblots remain in the source data. While reducing gels will show changes in size because\nof disruption of disulfide bonds, non-reducing gels maintain the intact protein structure\nand therefore identify whether or not there is translation of the full protein size. As\nshown in Fig.3b (updated to a white background to match the style in rest of Western\nblot figures), in non-reducing western blots of WT and KO hNSC using anti-BAI1\nantibody, WT cells show a primary band at 174kDa and two lighter bands below, and\nKO cells show elimination of the 174kDa band, with two barely visible bands below. The\nbands that appear below 174 kDa may reflect either post-translational modifications\n[PMID 38941066], or minor non-specific antibody binding under these conditions.\nHowever, most critically, the 174kDa full length protein band is completely absent in KO\ncells. Importantly, the consistency of the actin loading control across all samples rules\nout differences in protein loading or transfer efficiency in detection. We would also\nhighlight that this human BAI1 antibody (AF4969; R&D systems) in Fig. 3b recognizes\nthe 174kDa BAI1 band in BAI1 overexpression HEK293T cell lysates (+) (Response\nletter_Fig. 2). Note that in BAI1 overexpressing HEK293T cells, full-length BAI1 is\nobserved at two molecular weights due to post translational modifications, however, the\nmanufacturer reports <2% cross-reactivity with recombinant human BAI3 (170-130 kDa\nmolecular weight). We have added the empty vector (-) negative HEK293T cell control\nlysates into the Supplementary Fig.3a.\n3\n+ -\nThird at the functional-level, as shown in Fig. 3c-d, we further\nauthenticate our BAI1 KO cells by performing PLA specific for\nthe protein-protein interaction of BAI1 with its ligand, C1q, in both 260 kDa —\nBAI1 KO and WT cells. PLA is capable of detecting protein-\nprotein interactions with high specificity by generating a\n160 kDa —\nfluorescent signal only when antibodies bound to target proteins\nare in close proximity. In these experiments, our results clearly\ndemonstrate the absence of fluorescent signal only in BAI1 KO\ncells upon C1q stimulation -- while in BAI1 WT cells, upon C1q\nstimulation, a significant increase in fluorescent signal is Response letter_Fig.\nproduced. These BAI1-C1q PLA results visually demonstrate 2. BAI1 band in BAI1\nthat our BAI1 KO cells exhibit loss of BAI1 protein and function, overexpression\nfurther corroborating that BAI1 is fully deleted at the protein level HEK293T control (+)\nin our BAI1 KO cell line. vs. HEK293T empty\nvector negative\nTaken together, these genetic, proteomic, and functional data control (-) lysate.\nsupport that BAI1 expression has been effectively deleted in KO\nhNSC\nWe have updated the Figure legends, Methods, and Online Methods to include the\nadditional information as shown below:\n“Fig.3: b, Loss of full-length 174kDa BAI1 protein in BAI1 KO vs. WT hNSC\nline visualized by Western blot.”\n“Supplementary Fig.3: a, Commercial BAI1 antibodies generated using\npeptides from C-terminal vs. N-terminal region recognize full-length 174kDa\nBAI1 protein when analyzed using BAI1 overexpression lysate (+) vs.\nempty vector negative HEK293T control lysate (-) (Novus Biologicals)\nvia Western blot under reducing conditions.”\n“Methods: CRISPR Cas9 hNSC BAI1 genetic deletion\nPage 14, line 40 forward: Stable loss of total BAI1 protein (174 kDa)\nexpression was confirmed by Western blotting (Fig.3b), and functional-level,\nby performing PLA specific for the protein-protein interaction of BAI1 with its\nligand, C1q, in both BAI1 KO and WT cells (Fig.3c-d). Two different BAI1\nKO lines were generated with the same method. No substantial differences\nbetween the cell lines generated were detected regarding their CD133+\ncontent, multipotency, migration, or proliferative capacity. Follow-up\nexperiments in vitro were done at least independent experimental triplicates.\nBAI1 WT and BAI1 KO hNSC exhibited sustained normal karyotype\n(analyzed by Cell Line Genetics), high CD133+ stem cell proportions, stable\ngrowth rate under in vitro growth conditions, migration response, and\n4\nmultipotency in neural lineage differentiation (Supplementary Fig.2f-n)\ndemonstrating that both cell lines retained normal hNSC characteristics.”\n“Online Methods: BAI1 KO hNSC characterization\nPage 29, line 337 forward: Human BAI1 antibody (R&D systems, AF4969)\nused for analyzing total BAI1 protein in BAI1 WT vs. KO hNSC in non-\nreducing Western blots (Fig. 3b) recognizes the correct BAI1 band (174\nkDa) in BAI1 overexpression HEK293T cell lysate (+) (Novus Biologicals,\nNBP2-08183) (Supplementary Fig. 3a). In BAI1 overexpressing HEK293T\ncells, full-length BAI1 is present at two different molecular weights due to\npost translational modifications. Manufacturer of the antibody reports <2%\ncross-reactivity with recombinant human BAI3 at 170-130 kDa molecular\nweight.”\n3. For the experiments with the BAI1 neutralizing antibody (nAb) shown in Fig. 6, it is\nunclear which BAI1 antibody was used by the authors for these experiments. Also, how\nwas this antibody characterized? How do the authors know that it is truly a neutralizing\nantibody? Obviously, it is possible for antibodies to bind to a receptor but not neutralize\nligand binding, and thus it is important for the authors to provide more information about\nthe characterization of this antibody. The authors provide a citation for a previous\ncharacterization of the C1q nAb that was used in these studies, but no comparable\ncitation is provided for the BAI1 nAb.\nThank you for bringing this to our attention. For BAI1 neutralization we used Human\nBAI1 Antibody (Fig. 6a; R&D systems, AF4969). Data from the manufacturer\ndemonstrates the capacity of the BAI1 antibody to neutralize the the enhancement of\nadhesion of BCE C/D1b bovine cornea derived endothelial cells induced by BAI-1 at a\ndose of 2-8 µg/mL in the presence of 0.5 µg/mL Recombinant Human BAI-1.\nAdditionally, characterization of cross reactivity in direct ELISAs identifies less than 2%\ncross-reactivity with recombinant human BAI3, supporting the specificity of this effect.\nWe have added this information about the antibody in the Results Page 7, Line 7,\nMethods Page 11, Line 15, and the Online Methods Key Resources Table.\nReviewer #2 (Remarks to the Author):\nReview of the manuscript entitled “C1q drives neural stem cell quiescence by regulating\ncell cycle and metabolism through BAI1”.\nIn this manuscript, the authors describe a role for the complement-derived pattern\nrecognition molecule C1q in neural stem cell (NSC) quiescence. The team had\npreviously identified several new receptors for C1q, including brain angiogenesis\ninhibitor (BAI1). Here, they follow up this functional interaction and demonstrate C1q\nbinding to BAI1 on NSCs induces, at minimum, two events: firstly, BAI1-mediated\nsequestering of MDM2 which, in turn, increases p53 phosphorylation and blocks cell\ncycle progression an secondly, BAI1-depenent internalization of C1q which enables\ninteraction of C1q with the mitochondrial C1q receptor p32. The latter than induces a\n5\nshift from OXPHOS to aerobic glycolysis which also supports quiescence. As a general\nmodel, the authors argue that age- or inflammation/trauma induced increases in local\nC1q production may sustain NSC quiescence and inhibit activation when desired\n(during tissue repair, etc.).\nThe team makes its case using a set of elegant and detailed in vitro experiments and\nargues that this pathway is important in vivo by showing that the engraftment of BAI-KO\nhuman NSC into the brains of mice exposed to an acute CNS injury model (which is\nassociated with local increases in C1q) rescues the decline in hNSC proliferation when\ncompared to WT hNSC.\nOverall, the study is interesting and timely and its strength lays in the fact that it further\nexplored a new C1q-receptor interaction. Given the emerging central role for C1q\nacross human disease and specifically in CNS pathology settings, C1q is becoming a\nvaluable therapeutic target. The large majority in the field, however, focusses on the\n‘canonical’ C1q-repetior interactions (gC1qR and cC1qR). C1q biology is highly\ncomplex, and it is therefore paramount to better understand its biology for successful\nfuture targeted interventions. This leads to the second strength of the work: the authors\nargue that the effects of C1q in each tissue will depend on the C1q receptor ‘availability’\nwhich then also dictates the specific outcome of C1q activity. This aligns well with the\nemerging realization that local complement impact is highly cell and context specific.\nThere are several areas where the work can (and needs to be improved):\n1. A major issue is that the authors have not satisfactorily dissected the effects on\nproliferation, self-renewal, cell death and survival. Particularly Extended data Figure 3B\nand C is difficult to reconcile as the control phenotype seems to be driven by a single\ncell that divides extensively.\nWe agree that understanding the complex biology of C1q biology important for future\ntargeted interventions, and that these data combined with our previous description of\nC1q receptors provide insight the cell and context specific impacts of C1q. Indeed, we\nhave several followup manuscripts addressing this in preparation, including one that\nfocuses on the role of C1q in decline of neurogenesis in aging brain.\nIn interpreting this comment, we feel that we may have failed to provide sufficient clarity\nin the figure legends and methods to ensure that the data presented can be fully\ninterpreted. In the response below, we address the Reviewer’s concern regarding\nSupplementary Fig.3b and c and the contribution of a single cell to the control\nphenotype, as well as the underpinnings of how proliferation, self-renewal, cell death\nand survival.\nFirst, we would like to note that Supplementary Fig. 3b,c shows supporting schematic\nexamples for single cell Imagestream multispectral imaging flow cytometry gating for\nligand internalization (Supplementary Fig.3b), receptor-ligand intracellular co-\nlocalization (Supplementary Fig.3c) and spot counts using IDEAS software, relevant for\nImagestream data in Fig.4 and Fig.5. Accordingly, we believe the reviewer intended\n6\nto refer to Supplementary Fig.1d,e in the revised manuscript, which is an example\nof the paradigm employed for quantification of cell death in the control vs. C1q-treated\nFucci mNSC during fluorescent live-cell imaging (Fig.1j).\nIn Supplemenatry Fig.1d,e, the 30 cells shown for each group represent a single\nbiological replicate. However, a total of n=4 independent experiments (n=120 cells/\ngroup) were used to generate the data shown in the main figure (Fig. 1j), making the\nimpact of any single cell on the final analysis quite small. In addition, it is important to\nconsider what that impact would be; removing the cell in question would only serve to\nincrease the proportion of dying cells in the control condition, thereby further enhancing\nthe observed effect of C1q. Finally, as shown in Supplementary Fig.1a,b in the revised\nmanuscript, we also report quantification of the effect of C1q at the same timepoint via\nimmunocytochemical analysis of cleaved caspase-3 (CC3) staining, again conducted\nacross 3 independent experiments, providing orthogonal validation of this result.\nIn addition to clarifying that number of replicates and cells quantified in Supplementary\nFig. 1d,e are sufficient to enable interpretation, we have sought to clarify this in parallel\nfor the other experiments that dissect the effects of C1q on proliferation, self-renewal,\ncell death and survival. As described in the manuscript, we performed a combination of\nanalyses including BrdU/Edu proliferation assays, RealTime-Glo Annexin V multiplex\nassays, live cell imaging of Fucci-mNSC for cell cycle phases in subsequent timepoints\nand cell tracking for cell death, CC3 immunostaining, clonal neurosphere assays,\nRNAseq pathway enrichment analysis of quiescence, activation, and neural\ndifferentiation associated transcripts, and Western blot analysis of total protein for\nundifferentiated NSC/progenitor markers vs. lineage commitment markers. In addition,\nthe effects of C1q on NSC were also tested after short-term priming (up to 48h;\nproliferation) and long-term priming (2, 4, 6, or 8 weeks; proliferation, self-renewal, cell\ndeath, differentiation) to model prolonged C1q elevation caused by CNS injury/aging in\norder to dissect the effects of C1q on proliferation, self-renewal, cell death and survival.\nAccordingly, we have updated the Figure legends and Methods to include additional\ninformation as shown below:\n“Fig 1.h-i, Fluorescent live-cell imaging-based quantification of G0/G1\n(non-fluorescent/Cdt1-red) (h), or G1/S, S, G2 and M (Geminin-green) (i)\ncell cycle phase proportions in C1q-treated Fucci-mNSC vs. controls (n = 4\nindependent experiments). Data represent 150 cells plated/group per\nexperiment, total n=600 cells plated/group based on quantification of all\ncells in each microwell at each timepoint. j, Survival of C1q-treated Fucci-\nmNSC during live-cell fluorescence imaging vs. controls quantified using\nsingle-cell tracking of randomly selected cells from 0d to 7d (n = 4\nindependent experiments, 30 cells/group per experiment, total n=120 cells/\ngroup; tracking example in Supplementary Figs.1d,e).”\n“Supplementary Fig.1: d-e, A representative pedigree map of Fucci-mNSC\nlive cell fluorescence imaging, in which 30 cells in G1 phase were\nrandomly selected for tracking at 0h in each group. This is a graphic\n7\nexample the data collection that was used for Fig.1j, in which n=4\nindependent experiments with 30 cells per replicate/group (a total of 120\ncells per group) were used to test for differences in viability, comparing\nnon-treated control (d) vs. C1q [200nM]-treated Fucci-mNSC over the\ncourse of a 7-day experiment (e). 30 cells in G1 phase were randomly\nselected for tracking at 0h in each group per independent biological\nexperiment.”\n“Methods: Page 11, Fluorescence live cell imaging and tracking\nFucci-mNSC were plated into custom-made PDMS microwells at a\ndensity of 150 cells/well without (non-treated control) or with 0.1nM or\n200nM C1q in DM, imaged using Olympus Vivaview® fluorescence\nincubator microscope at 20x objective in 20min intervals for 7 days. Image\nanalysis was performed using Imaris version 7.5.2. from image montages\nconsisting of 3x3 stitched images as previously described23. For each\nindependent experiment five technical replicate microwells were seeded,\nand one microwell with comparable cell confluences per group was\ncaptured and analyzed. The total number of Fucci-mNSC and relative\nproportions of the cells in G1 or S, G2, or M phases in each timepoint per\nwell were quantified from the image montages at 0h, 48h, 72h, 96h, and\n7d during in vitro culture (Fig.1g-i, Supplementary Fig.1c).\nFor single-cell tracking of NSC survival (Fig. 1j, Supplementary\nFig.1d,e), 30 Fucci-mNSC (all in G1 within the first frame of the movie\ncorresponding to 0h timepoint) were randomly selected per each\nindependent experiment to be tracked frame-by-frame for up to 7 days\nusing the manual tracking feature of Imaris. Cell death of the\ntracked Fucci-mNSC is shown as an average percentage of dead cells\nrelative total number of tracked cells (Fig. 1j). An example of the paradigm\nemployed for quantification of viability in the Fucci-mNSC (Supplementary\nFig.1d,e) represents a single independent experiment; 30 cells for each\ngroup. A total of 4 independent experiments (Total n=120 cells/group) was\nused to generate the data shown in Fig. 1j. All analyses were performed\nby investigators blinded to experimental conditions/groups.”\n“Online Methods: Page 28, Quantification of cell cycle phases and cell\nsurvival during fluorescence live cell imaging\nFor cell cycle-, single cell division, death analysis, Fucci-mNSC\nwere plated into custom-made PDMS microwells at a density of 150 cells/\nwell without (non-treated control) or with 0.1nM or 200nM C1q in DM. For\neach independent experiment, five technical replicate microwells were\nseeded, and one microwell with comparable cell confluences per group\nwas captured and analyzed. The total number of Fucci-mNSC and relative\nproportions of the cells in G1 or S, G2, or M phases per microwell in each\ntimepoint were quantified from the image montages at 0h, 48h, 72h, 96h,\nand 7d during in vitro culture (Fig.1g-i, Supplementary Fig.1c). The data of\n8\neach independent experiment was collected from a single microwell on\nseparate petri-dishes, that were either imaged separately (fluorescence\ntime-lapse) or simultaneously (DIC-only time-lapse). A total of 4\nindependent experiments (n=150 cells plated for each group per each\nreplicate) was used to generate the data shown in the main figure (Fig.1g-\ni, Supplementary Fig.1c) based on quantification of all cells in each\nmicrowell at each timepoint; (Total n= 600 cells plated/group).\nFor single-cell tracking of NSC survival (Fig.1j, Supplementary\nFig.1d,e), we randomly selected 30 Fucci-mNSC (all in G1 within the first\nframe of the movie corresponding to 0h timepoint) per each independent\nexperiment to be tracked frame-by-frame using the manual tracking\nfeature of Imaris for up to 7 days; tracking experiments included the\nprogeny of all selected parent cells. In each frame, a visually determined\nand manually selected physical center of the cell was used as a reference\npoint for cell tracking. After cell division, both daughter cells were tracked\nuntil the end of the experiment unless the cell died or was lost to\nidentification. An example of the paradigm employed for quantification of\nviability in the Fucci mNSC (Supplementary Fig.1d,e) represents a single\nindependent experiment; 30 cells for each group.”\n2. Although the authors likely want to make it easier ‘on the eye’ by showing the control\ndata as a 100% threshold line, raw data are preferred to see the spread here.\nWe have included the Source data files with raw data per each independent experiment\ninto this submission.\n3. In general, all experiment dealing with phosphorylated proteins (p53 etc.) need to\nshow total protein as comparator).\nWe thank the reviewer for raising this point. We have revised the Western blot data in\nFig.2b and Fig.3i in the resubmitted manuscript accordingly; total p53Ser15 protein are\nnow normalized to total p53 protein.\nSee also edits in “Methods: Page 12, line 33 ..anti-Phospho-p53 (Ser15)\n(1:300, Cell Signaling, 16G8, 9286), anti-p53 (1:500, Cell Signaling,\n9282s)” and “Page 13, line 2: Phospho-p53 (Ser15) bands were\nnormalized to total p53. “\n“Fig.2: Blood plasma C1q concentration activates p53 signal transduction\ndriving cell cycle quiescence. b, Western blot analysis of phospho-\np53Ser15 in hNSC treated with C1q [200nM] vs. non-treated or heat-\ninactivated C1q [200nM] (T°Inact 200nM) negative controls. Optical band\nintensities were normalized to p53 and shown relative to non-treated\ncontrols (n = 4 independent experiments).”\n9\n“Fig.3: C1q-driven p53 signal transduction is BAI1-dependent. g, Western\nblot analysis of total phospho-p53Ser15 in C1q-treated BAI1 WT or KO\nhNSC relative to each non-treated control (ctrl, dashed line) (n = 4-5\nindependent experiments). Optical band densities were normalized to total\np53.”\n4. The mitochondrial staining for C1q/p32 etc. are not convincing and should be\nrepeated in high resolution (like Figure 5I, which is very nice).\nWe thank the reviewer for bringing this to our attention.\nWe have interpreted this comment as best we can. We do not show mitochondrial\nstaining for C1q/p32 in the original manuscript. We show mitochondrial staining in Fig 5I\n(now 5L), which is mitotracker at high resolution to enable quantification of\nmitochondrial segmentation/morphology. Elsewhere, we show staining for DAPI,\nmitotracker, and phospho-p53 in Figure 3J, where the point of including mitotracker was\nto identify cytoplasmic versus nuclear (DAPI) area, in order to quantify the nuclear\ntranslocation of phospho-p53. We have clarified this in the resubmission, and removed\na graph of mitochondrial localization from this figure in deference to the reviewer’s\ncomment. We do not feel that this use of mitotracker necessitates re-imaging at higher\nresolution. We believe that the other relevant data to this comment is in Supplementary\nFig. 5e, which shows DAPI, mitochondrial marker TOM20, and p32 in C1q treated WT\nvs KO hNSC, with the principal goal to quantify p32 localization in the mitochondria.\nAgain, we believe that because mitochondrial morphology is not analyzed here, the\nresolution is sufficient for this analysis.\nWe have revised the Resubmitted Fig.3 legend to correspond the data shown in\nthe graph:\n“C1q-driven p53 signal transduction is BAI1-dependent. j, Example\nimages of phospho-p53Ser15 staining in Dapi+ nucleus vs. cytoplasma with\nMitotracker+ mitochondria. k, Quantification of phospho-p53Ser15\nlocalization in nucleus in C1q-treated BAI1 WT or KO hNSC using ICC\nrelative to each controls (n = 3-4 independent experiments). Mean ± s.e.m,\nk 1-way ANOVA with post hoc test.”\n5. The authors argue that BAL1 induces C1q internalization which then can bind to p32\nas it may be released. However, there should be a high-resolution triple staining for\nC1q/BAL1/p32 (which should be negative) to support this model.\nThe principal hypothesis we intended to test was whether internalized C1q interacts with\nintracellular p32 in a BAI1-dependent manner, as shown by PLA (Fig.4v-x; Fig.5c-d). In\nreflecting on the Reviewer’s question, we realized that the schematic in Figure 5G\n(hypothesized metabolic outcome for BAI1-dependent intracellular C1q-p32 interaction\nand decreased p32 stability) was misleading because - although not intended - it\nimplied C1q release. As intracellular protein interactions and trafficking are areas where\n10\nmuch of the field of cell biology is seeking new understanding, the question of whether\nC1q is released from BAI1 within the endosomes remains to be determined and is\noutside of the scope of this submission. However, we appreciate the Reviewer’s point in\nrequesting direct evidence for interactions after internalization, and have sought to\nprovide this data. We have therefore added analyses of C1q-BAI1-p32 interactions\nusing pull-downs of purified human recombinant proteins in situ. These data\ndemonstrate direct interactions between C1q-BAI1, C1q-p32, and BAI1-p32, and that\nC1q-BAI1-p32 can be pulled down as a complex (Response letter_Fig. 3, Fig.5f). In\ndeference to the Reviewer’s points, we have also revised the cartoon in Fig.5g to show\nC1q-BAI1-p32 in complex, and added a line to the discussion addressing the\nuncertainty of how dynamic this complex might be within the cell.\nResponse letter_Fig. 3. Direct interaction between C1q-BAI1, C1q-p32,\nand BAI1-p32 tested using in situ pull-down of human purified or\nrecombinant proteins.\nWe have revised the Results, Figure legend, Discussion, and Methods\naccordingly:\n“Results, Page 6, lines 8-12: Pull-down of purified human proteins in situ\ndemonstrated a direct interaction between C1q-BAI1 and C1q-p32, as\nwell as BAI1-p32, and that C1q-BAI1-p32 can be pulled down as a\ncomplex (Fig.5f). Together, these data show that BAI1-mediates\nendocytosis of C1q enabling intracellular protein interactions between\nC1q-p32, BAI1-p32, or C1q-BAI1-p32, decreasing total p32.”\n“Fig.5: C1q-p32 intracellular interaction drives p32 instability and aerobic\nglycolysis in BAI1-dependent manner. f, Pull-down of human proteins in\nsitu demonstrates direct interaction between C1q-BAI1, C1q-p32, and\nBAI1-p32.”\n11\n“Discussion: Page 9, lines 3-6: In addition, our data demonstrates a direct\ninteraction not only between C1q-BAI1 and C1q-p32 but also BAI1-p32,\nenabling pulldown of C1q-BAI1-p32 as a complex. The role of BAI1-p32\ninteraction as well as how dynamic C1q-BAI1-p32 complex might be within\nthe cell remains uncertain.”\n.\n“Methods: Page 19, Pulldown protein interaction analysis\nDirect interaction between C1q, BAI1 and p32 in situ was tested by\npulldown assays using purified human proteins following the manufacturer\ninstructions. Briefly, 5 µg bait His tag recombinant human BAI1\n(SinoBiological, 4969-BA) was prepared in a 1x binding/wash buffer (50\nmM sodium phosphate, 300 mM NaCl, 0.01% Tween-20) and immobilized\non 2 mg magnetic beads (Dynabeads His-Tag Isolation and Pulldown,\nInvitrogen, 10103D) by incubation on a rocker for 10 min at RT. Tubes\ncontaining the bait protein immobilized on beads were placed on a magnet\n(Invitrogen, 12321D) and the supernatant containing excess bait protein\nwas collected for Western blot validation. Beads were washed four times\nwith 1x binding/wash buffer to remove any remaining excess bait protein.\n5 µg of prey proteins purified human C1q (MyBioSource, MBS147305) and\nrecombinant human p32 (LSBio, LS-G3375-20) were prepared in a 1x\npulldown buffer (3.25 mM sodium phosphate, 70 mM NaCl, 0.01%\nTween-20) and incubated with the immobilized bait protein on a rocker for\n15 min at RT. Prior to pulldown protein capture, tubes containing the\nimmobilized bait-prey protein complexes were placed on a magnet and\nwashed four times with 1x binding/wash buffer to remove any remaining\nunbound prey proteins. Bait protein and bound prey proteins were eluted\nusing his-elution buffer (300 mM imidazole, 50 mM sodium phosphate,\n300 mM NaCl, 0.005% Tween-20) and the eluted samples were analyzed\nusing Western blotting. The original blots are in the Source data.”\n6. Figure 1E: something is not right here with the last 2 conditions: why does the isotype\ncontrol IgG antibody further amplify the reducing effect of C1q on NSC proliferation and\nalso has such a strong inhibitory effect without C1q addition?\nWe thank the reviewer for pointing this out. The data in this figure (Fig.1f in the revised\nmanuscript) show that treatment of hNSC with C1q 200nM alone (grey bar) is not\ndifferent from treatment of hNSC with C1q 200nM + IgG isotope control (the color of this\nbar has been changed from blue to grey for clarity). However, the Reviewer’s comment\nled us to identify an error on this graph which resulted in some confusion; we greatly\nappreciate the opportunity to correct this issue. Specifically a single replicate\nexperiment in which cells were treated with isotype control IgG antibody alone (no C1q)\nwas accidentally included; critically, not only was this data from a single well, but\ntracking this data back in the experimental notes identified that the chamber slide well\nhad dried out during culture and should have been excluded altogether. We apologize\nfor this error, and have rechecked all of the data points for this figure to ensure no\nadditional errors are present. We have corrected Fig. 1f by removing this bar from the\n12\ngraph. Again, we would highlight that there is no significant difference between hNSC\ntreated with C1q 200nM vs. C1q 200nM + Isotype control IgG antibody groups,\ndemonstrating that the isotype control IgG does not amplify the C1q-driven decline in\nproliferation. This is the relevant experiment, because fetal brain-derived hNSC do not\nexpress C1q or downstream complement pathway components, thus the addition of\nexogenous C1q is necessary to assess any potential effect of the isotope control.\n7. Does ERK inhibition have similar effects on NSC proliferation?\nIn our initial report on the identification of multiple novel C1q interacting receptors, we\nshowed that the GPCR inhibitor pertussin toxin (PTX) blocked proliferation decline but\nnot migration (PMID: 32894219), supporting our hypothesis that C1q-mediated decline\nin hNSC proliferation is mediated by cell surface GPCR/s such as BAI1. However, to the\nreviewer's question, we also reported that Erk inhibition using a selective chemical\ninhibitor (PD98059) attenuated C1q [200 nM] driven changes in both migration and\nproliferation (PMID: 32894219), suggesting that the Erk1/2 pathway is involved in\nmultiple cellular functions in hNSC (PMID: 12892714, PMID: 24396730).\nOne would predict that C1q would engage multiple of the C1q receptors identified,\nsimultaneously activating multiple intracellular pathways depending on the relative\nbinding affinity of each receptor for C1q and therefore C1q concentration. Additionally,\nthree of these receptors are GPCRs, and GPCRs can also engage diverse signaling\nmolecules, thereby modulating not only the canonical cellular responses but also\nnoncanonical responses typically associated with activation of other cascades such as\nMAPK/ERK signaling (PMID: 32576977, PMID: 24396730, PMID: 29699693). In\nalignment with this anticipated complexity, we stated the following under the discussion:\n“Importantly, BAI1 may have other ligands that can also trigger quiescence in hNSC,\nand given that C1q internalization was only partially inhibited in BAI1 KO, other\ncandidate C1q receptors could play a role in C1q internalization (PMID: 11827788,\nPMID: 28827538, PMID: 36330337)”. To address the reviewer’s question, we propose\nto expand this element of the discussion to state:\nPage 8, lines 38-42, and Page 9, lines 1-3: “Importantly, while we have\npreviously shown that GPCR inhibition via pertussin toxin (PTX) blocked\nC1q-induced proliferation decline but not C1q-induced migration, Erk\ninhibition via PD98059 modulated both migration and proliferation. These\ndata suggest that cell surface GPCR/s such as BAI1 play a key role in C1q\nproliferation signaling, but also that the Erk1/2 pathway is involved in\nmultiple cellular functions in hNSC (PMID: 12892714, PMID: 24396730). In\naddition, BAI1 may have other ligands that can also trigger quiescence in\nhNSC, and given that C1q internalization was only partially inhibited in\nBAI1 KO, other candidate C1q receptors could play a role in C1q\ninternalization and modulation of cell proliferation”.\n13\n8. Figure 4K: the negative C3aR/C1q control looks\npositive (?) or has a very high background which\nmakes it difficult to interpret the other data –\nthoughts?\nThank you very much for pointing this out. It is clear\nthat we did not select the most representative\nexample. To allay any concerns in this regard, we\nhave included a selection of multiple raw image\nstream examples from both C1q-C3aR negative\ncontrols and from C1q-BAI1 positive condition for\npositive context (Response letter_Fig.4). Cell #227\nin the Response letter_Fig.3 is the original control\nshown in Fig. 4k, but when the brightness/contrast\nof the full figure was adjusted, we agree that the\noverlay could be misinterpreted. Accordingly, we\nhave replaced Fig. 4k with cell #425 from C1q-\nC3aR negative control in the Response letter_Fig.4.\nIn addition, we have edited the Methods, and Online\nMethods to include more details as follows:\nMethods, Page 16: “For imaging flow\ncytometry, hNSC were plated on PLO-\nlaminin-coated 75cm2 or 225cm2 cell culture\nflasks in GM. At 70-80% confluence, hNSC\nwere treated with 200nM C1q or an equal\nvolume of GM (non-treated control) for 5min,\n15min, 30min, 1h, 2h, or 24h followed by\nDPBS-/- wash and detachment using\nTrypsin/EDTA or non-enzymatic cell\ndissociation solution (Sigma-Aldrich,\nC5914). For matched ligand-G protein-coupled receptor C3a-C3aR complex\ninternalization analysis, hNSC were treated\nwith purified human C3a anaphylatoxin\n(Complement Technology, A118) at 100nM\nconcentration or an equal volume of GM\n(non-treated control) for 5min, 15min, or\n30min. For matched ligand-tyrosine kinase\nreceptor EGF-EGFR complex internalization\nanalysis, hNSC were treated with pHrodo™\nred Epidermal Growth Factor (EGF) Response letter_Fig.4: Multiple\nConjugate (Thermo Fisher Scientific,\nraw image stream examples\nP35374) at 40ng/mL concentration or an\nfrom both C1q-C3aR negative\nequal volume of GM (non-treated control) for\ncontrols and from C1q-BAI1\npositive condition.\n14\n3min. For testing C1q colocalization in endosomes or Golgi, hNSC were\ntransfected with either CellLight™ Early Endosomes-RFP, BacMam 2.0\n(Invitrogen, C10587), or CellLight™ Golgi-RFP, BacMam 2.0 (Invitrogen,\nC10593). The following morning, the cells were washed and treated with\n200nM C1q for 24h. Cells were aliquoted and fixed using 2% PFA for\n15min on ice followed by wash with 1% fish gelatin in 0.1M TBS,\nblocking, and 1min permeabilization using DPBS supplemented with 0.1%\nor 0.02% Triton X-100, 5% goat or donkey serum. Primary antibodies\ndiluted in 0.1M TBS wash buffer with 1% fish gelatin and 5% serum were\nincubated with cells overnight at +4°C. Primary antibodies used were\nanti-C1q (1:50, Abcam, Ab71940), anti-human BAI1 (1:50, R&D systems,\nAF4969), anti-BAI1 (1:50, Lifespan Biosciences, LS-C120632), anti-human\ncomplement C3a (1:50, Millipore/Chemicon, CBL191), anti-C3aR (1:50,\nAbcam, Ab103629), anti-EGFR (Abcam, EP38y, ab52894), recombinant\nanti-GC1qR (1:50, Abcam, Ab24733) followed by 30min incubation RT with\n1:500 dilution of secondary antibodies conjugated with 555, 488, or 647\nfluorochromes (Invitrogen, A131570, A312572, A21202, A21206, A21141,\nA31571, A31573, A21240 or A21447). The BAI1 antibodies used for\nWestern blotting, PLA, or imaging flow cytometry all recognize the full\nlength BAI1 when evaluated using BAI1 over expression lysate and empty\nvector negative HEK293T control lysate (Novus Biologicals;\nSupplementary Fig.3a). Single cells at a density of 20,000 cells/µl were\nimaged using Amnis Imagestream x Mark II Imaging Flow Cytometer and\nanalyzed using IDEAS version 6.2 according to the manufacturer\ninstructions (Cytek Biosciences)108. Gating were set using unstained\nnegative control cells, secondary antibody only controls, single labeled\ncells, and antibody compensation beads (Life technologies, A10513). For\na gating examples see also Supplementary Fig.3. For protein\ninternalization, score ≥0.5 was considered internalized. Time course data\nwas collected from one to four independent experiments with one sample\nper each. Each independent experiment consists of one flask treated for\nindependent times or plated and treated on independent days.”\n“Online Methods: Page 30, Ligand/receptor internalization analysis using\nimaging flow cytometry\nSingle cells at a density of 20,000 cells/µl in 0.1M TBS wash buffer\nwith 1% fish gelatin were imaged using Amnis Imagestream x Mark II\nImaging Flow Cytometer and analyzed using IDEAS software version 6.2\naccording to the manufacturer instructions (Cytek Biosciences)102. Briefly,\nfor ligand/receptor internalization analysis cells were eroded using an\neroding mask (Fig.4e) of 7-pixel (Fig.4b,c,o-q, and Supplementary\nFig.4b,c,f,g, o) or 3-pixel on a brightfield channel (Fig.4d,f and Fig.4u, and\nFig.5 a,b). Cells with a ligand/receptor internalization score ≥0.5 was\ngated as internalized (Supplementary Fig.3b, Supplementary Fig. 4c,g,o).\nFor colocalization analysis, cell population exhibiting ligand and receptor\n15\ninternalization were plotted on a histogram using bright detail similarity R3\nfeature designed to specifically to compare the small bright image detail of\ntwo images. This feature is the log transformed Pearson’s correlation\ncoefficient of the localized bright spots with a radius of 3 pixels or less\nwithin the masked area in the two input images. Since the bright spots in\nthe two images are either correlated (in the same spatial location) or\nuncorrelated (in different spatial locations), the correlation coefficient\nvaries between 0 (uncorrelated) and 1 (perfect correlation) and does not\nassume negative values. The coefficient is log transformed to increase the\ndynamic range between (0, infinity). In this study, mean bright detail\nsimilarity R3 score (co-localization score) ≥1.5 was considered colocalized\n(Fig.4d,o,u, and Supplementary Figs.3c and 4d,h). Spot count feature\n(Supplementary Fig.4o) was used for measuring the quantity of\ninternalized protein per cell (Fig.4q, Fig.5b, Supplementary Fig.3c).”\n“Fig.4: Endocytic internalization of C1q-BAI1 complex allows intracellular\nC1q-p32 interaction and p32 instability.\na-c,e, Timecourse Imagestream analysis of C1q internalization kinetics in\nimmunolabeled C1q [200 nM]-treated and non-treated control hNSC (a-c)\nusing 7-pixel component masking visualized in e (n = 2-5 independent\nexperiments per timepoint). Cells with a ligand internalization score ³0.5\nwere gated as internalized. d,f-g, Co-localization of intracellular C1q\nimmunolabel and Rab5-RFP reporter transfected hNSC endosomes (d,f)\nvs. Golgi-RFP (g) with 3-pixel component masking. Co-localization score\n(bright detail similarity R3 score) ≥ 1.5 was considered colocalized….j-k,\nIntracellular ligand-receptor co-localization after immunolabeling of\nmatched ligand-receptor positive control C3a-C3aR (j) vs. mis-matched\nnegative control C1q-C3aR (k). l,n-o, Timecourse analysis of C1q-BAI1\ncomplex internalization kinetics in BAI1+ in-focus single cells using\ncomponent masking (n = 2-3 independent experiments per timepoint). p-q,\nBAI1 KO decreases the proportion of in-focus single hNSC with\nintracellular C1q (p) and the overall quantity of internalized C1q per cell\nwithin this sub-population analyzed (q) relative to C1q [200 nM]-treated\nBAI1 WT (ctrl, dashed line) (n = 3 independent experiments).”\n“Supplementary Fig.4: C1q-bait-intracellular prey-protein pull-down data\nand Imagestream analysis validation. Related to Fig.4 and 5. ….. b-e,\nTimecourse analysis of matched ligand-G protein-coupled receptor C3a-\nC3aR complex internalization kinetics in C3a+ in-focus single cells post-\nC3a treatment. f-j, Matched ligand-tyrosine kinase receptor EGF-EGFR\ncomplex internalization in EGF+ in-focus single cells post-phRodo EGF-\ntreatment in hNSC. An Imagestream example of pHrodo-EGF and EGFR\nICC staining (j). For ligand/receptor internalization analysis cells were\neroded using an eroding mask of 7-pixel on a brightfield channel and cells\nwith a ligand/receptor internalization score ≥0.5 were gated as\ninternalized. k-l, Timecourse Imagestream images of C1q-BAI1 complex\n16\ninternalization kinetics in BAI1+ in-focus single cells 15min (k) and 1h\npost-C1q treatment in BAI1 WT hNSC (l). m-n, C1q-BAI1 complex\ninternalization kinetics in-focus single cells 1h post-C1q treatment in BAI1\nKO hNSC (m), and example images of negative control background in\nnon-treated, C1q-stained hNSC vs. C1q-treated, 2. ab only-stained hNSC\n(n). o, An example of Imagestream component masking for spot counts in\nthe intracellular compartment to compare intracellular C1q quantity. p,\nImagestream analysis of p32 cellular localization and mean internalization\nscore in non-stained vs. p32 stained hNSC based on IDEAS software.”\n9. Figure 7: the analysis of C1q is very nice – why did the authors not perform the same\nanalysis for BAI1? It would be nice to see the expression profile of this C1q receptor in\nrelation to its ligand. It would have also added some suggestions to whether the\ninteraction by be autocrine or paracrine.\nThis is an interesting idea, unfortunately, in our hands, transcriptomic analysis is a poor\nway to look at the expression of most of the C1q receptors we have identified. This is\nparticularly true for the GPCRs, which are highly stable proteins in the cell membrane;\nas a result, transcription is maintained a level that is low or even undetectable in\nRNAseq.\nHowever, as per the Reviewer’s suggestion, we analyzed local Bai1 mRNA expression\nin acute SCI by different cell types using published scRNAseq data (PMID: 34132743).\nscRNAseq shows low Bai1 reads mostly detectable in spinal cord neural cell\npopulations, such as astrocytes, OPC/oligodendrocytes, ependymal, and neurons, and\nsome immune cells (R_Fig. 4A), and suggests that SCI does not have a large impact on\nBai1 expression (R_Fig. 4C). In contrast, C1qa in this scRNAseq dataset is highly\nexpressed by immune cells in both intact and injured spinal cord (R_Fig. 4C), and\nnearly all immune cell populations and spinal cord neural cell types upregulate C1qa\nafter SCI (R_Fig. 4D). To the Reviewer’s point, these data suggest a predominantly\nparacrine signaling mechanism for C1q-BAI1 interaction in spinal cord cells during\nhomeostasis and after injury (1dpi). However, subacute upregulation of C1qa reads\npost-SCI (3-7dpi) may also allow C1q-BAI1 autocrine interactions.\nWe are currently at max capacity for Supplementary Figures, however, if the Editor\nallows, we are more than happy to include the Response letter_Fig. 5 and this\ndiscussion into our MS as a Supplementary Figure.\nA Cell Types\nN M e o u n tr o o c p y h te il Bai1 Bai1 Bai1 Bai1\nMacrophage\nDendritic\nMicroglia\nDividing Myeloid\nFibroblast\nEndothelian\nPericyte\nOPC\nOligodendrocyte\nAstrocyte\nEpendymal\nNeuron\nLymphocyte\nIntact 1dpi 3dpi 7dpi\nCell Types\nB N M e o u n tr o o c p y h te il C1qa C1qa C1qa C1qa\nMacrophage\nDendritic\nMicroglia\nDividing Myeloid\nFibroblast\nEndothelian\nPericyte\nOPC\nOligodendrocyte\nAstrocyte\nEpendymal\nNeuron\nLymphocyte\nC D\nBai1 transcripts C1qa transcripts\nIntact 1dpi 3dpi 7dpi Intact 1dpi 3dpi 7dpi\nNeutrophil % Detected Neutrophil % Detected\nMonocyte 100 Monocyte 100\nMacrophage 75 Macrophage 75\nDendritic 50 Dendritic 50\nMicroglia 2 0 5 Microglia 2 0 5\nDividing Myeloid Dividing Myeloid\nFibroblast Log- Fibroblast Log-\nEndothelian Normalized Endothelian Normalized\nPericyte counts Pericyte counts\nOPC 1 OPC 4.6\nOligodendrocyte Oligodendrocyte\nAstrocyte Astrocyte\nEpendymal Ependymal\nNeuron 0 Neuron 0\nLymphocyte Lymphocyte\nResponse letter_Fig. 5 C1qa-Bai1 expression in spinal cord cell types in\niRnt_aFcitg .o r4 sCp1inqaal- Bcoari1d einxjpurreesds miono uins es.p ain-bal, cUoMrdA cPe lpl ltoytp oefs einx pinretascsti oonr ospf iBnaai 1\nvcso.r dC i1nqjuar edin mmoaujsoer. sap-bin, aUl McoArPd pcloetll opf oepxuplaretisosniosn boafs Beda i1o nv s.s cCR1NqaA sien q\n(mPaMID: 34132743). cLlo wpr oepu ladtiso nosf Bbaasi1e da roen m scaRinNlyA dseetqe c(PtaMbIleD :i n3 4s1p3in2a7l 4c3o)r. d\nnLeouwr arl eacdesll opfo pBualia1t ioanrse, msouscthly adse teacstatrbolcey tiens , spOinPaCl /cooligrdo dneenudrraol cycteelsl ,\neppoepnudlaytmionasl,, asnudch naesu raosntrso, caytneds , sOomPCe/ oimligmoduenned rcoeclylste (sa, )e. pCe1nqdaym isa l,h aignhdl y\nenxepurreosnsse, da bnyd imsommuen ei mcemllus nine bcoetlhls in(taa)c. t Can1dq ain jiusr edh gshplyin ael xcporreds s(bed). cb-yd ,\nDimomt pulnoet ocfe allsb uinn dbaontche inotfa Bcta ia1n (dc )in ojur rCed1 qsap i(nda)l tcraonrds c(rbip)t. sc p-de,r Dceoltl tpylpoet so fi n\nianbtaucntd oarn cinej uorfe dB asip1i n(acl) coor rdC 1(PqaM I(Dd): 3tr4a1n3sc2r7pips t3s) .p Eera cche lld toytp seisz ein r einptraecste onrt s\npinejrucreendt agsep inoaf lt hceo drde te(cPteMdI Dtr:a 3n4s1cr3ip2t7s4, 3w).h ileE athceh cdoolot r sinizteen sriteyp irnedsiecnattes s\nlpoegr-cneonrtmagaeliz oefd thcoeu dnetste pceterd c etrlal ntyspceri.p tSsC, Iw dhoilee st hneo tc hoalovre i nat ednrsaimtya itnicd iicmapteasc t\nolong B-naoir1m eaxlipzreeds scioounn (tcs) .p Nere caerllyl taylpl eim. SmCuIn deo ceesl l npootp hualavteio an sg raenadt i mnepuarcatl ocne ll\ntByapie1s euxppreregsusliaotne C(c1)q. aH aofwteerv SerC, I n(dea).r lTyh aelsl ei mdamtau nheig cheligll hpt othpeu laliktieolnihso oadn do f\nparacrine signaling mechanism may be prevalent for C1q-BAI1 interaction\nacutely 1dpi, however, subacute upregulation of C1qa reads post-SCI may\nin spinal cord neural progenitors during homeostasis and acutely 1dpi.\nalso allow C1q-BAI1 autocrine interactions.\nHowever, subacute upregulation of C1qa reads post-SCI may also allow\nC1q-BAI1 autocrine interactions in spinal cord neural cell lineages.\n18\n10. The in vivo experiment in Figure 7 solidifies the role of BAL1 in hNSC proliferation\ncontrol. It does not conclusively allow to associate this with the C1q activity though.\nCould the authors approach this somehow? For example, could there be a bulk RNA-\nseq analysis of in vitro WT and BAL1 KO hNST +/- C1q (as in Figure 6) to generate\nC1q-BAL1 axis (normal/perturbation) signatures and then utilize these signatures to\nassess the bulk RNA-seq signatures from re-isolated SC121-Brdu+ cells WT/KO cells in\nthe lesions (Figure 7D)?\nWe appreciate the reviewers thoughts on this matter, as we have found this issue\nchallenging to approach. We have previously attempted to re-isolate engrafted hNSC\n(PMID: 28479305). However, because these cells begin to migrate and differentiate\nquickly after transplantation (PMID: 24936450), the yield even at 2 weeks post-\ntransplantation is incredibly low. For example, 900 cells pooled from 2-5 transplanted\nanimals, out of 50,000 cells total transplanted (PMID: 28479305). We feel therefore that\nthis approach would not give an unbiased or reproducible sample from which to conduct\na comparison. While we absolutely agree in principle that an ideal experiment would\ndefinitely tie in vitro definition with mechanism to in vivo action, we do not see a path to\novercome this hurdle. For this reason, we have also a section in the discussion noting\nthat there may be other BAI1 ligands present in vivo that modulate the mechanism we\ndescribe in vitro.\n11. The manuscript has many errors when it comes to labeling and cross-referencing\nfigures and data. For example, the table is references erroneously as Extended Data 2,\nthere is no reference to Extended Data 3 in the text and Extended Data 8 (mentioned in\nthe text) are entirely missing, Extended Data 7 legend seems not to fit (ROS), etc..\nOverall, the structure of text, figures, and Extended data can be improved – it was at\ntimes difficult to navigate text and figures.\nWe apologize, and thank the reviewer sincerely for highlighting for us so that we could\naddress these errors; We have corrected the numbering of the Supplementary Figures.\nReviewer #3 (Remarks to the Author):\nIn this work the authors focus on the complement factor C1q and its effects on\nproliferation and quiescence of human neural stem cells (hNSCs). First, they\ndemonstrate that C1q treatment leads to these cells proliferating significantly less with\nthe absence of any changes in apoptosis, suggesting an induction of quiescence. Next,\nthey undertake a variety of studies to assess the underlying mechanism of this effect.\nThe first of these mechanisms is shown through changes in p53 phosphorylation and\nlocalization to the nucleus which seems to occur through MDM2 initiated by binding to\nBAI1. They then assess the internalization of C1q and suggest that this occurs via\nendocytosis. The authors also detail a novel role for C1q in mitochondrial changes in\nhNSCs acting through p32 and BAI1. Finally, the authors show that loss of BAI1\nrescues the C1q impairments in proliferation and that transplantation of BAI1 KO cells\nare more highly proliferative in a spinal cord injury model. These data are exciting and\n19\nexpand our understanding of the role of immune molecules in the control of proliferation\nand quiescence of neural stem cell populations. While I am enthusiastic about these\nstudies, I do have a number of criticisms that the authors should consider.\n-In Figure 1E, it is shown that IgG nab both alone or in the presence of C1q leads to a\n~5-fold larger reduction in BrdU positive cells than the when C1q is used alone. How\ncan this be as this is a control antibody?\nWe thank the reviewer for pointing this out. The data in this figure (Fig.1f in the revised\nmanuscript) show that treatment of hNSC with C1q 200nM alone (grey bar) is not\ndifferent from treatment of hNSC with C1q 200nM + IgG isotope control (the color of this\nbar has been changed from blue to grey for clarity). However, the Reviewer’s comment\nled us to identify an error on this graph which resulted in some confusion; we greatly\nappreciate the opportunity to correct this issue. Specifically a single replicate\nexperiment in which cells were treated with isotype control IgG antibody alone (no C1q)\nwas accidentally included; critically, not only was this data from a single well, but\ntracking this data back in the experimental notes identified that the chamber slide well\nhad dried out during culture and should have been excluded altogether. We apologize\nfor this error, and have rechecked all of the data points for this figure to ensure no\nadditional errors are present. We have corrected Fig. 1f by removing this bar from the\ngraph. Again, we would highlight that there is no significant difference between hNSC\ntreated with C1q 200nM vs. C1q 200nM + Isotype control IgG antibody groups,\ndemonstrating that the isotype control IgG does not amplify the C1q-driven decline in\nproliferation. This is the relevant experiment, because fetal brain-derived hNSC do not\nexpress C1q or downstream complement pathway components, thus the addition of\nexogenous C1q is necessary to assess any potential effect of the isotope control.\n…Moreover, in this same figure it’s shown that after priming for 4-8 weeks this actually\nleads to greater proliferation (ie. BrdU+ cells) than control. Can the authors explain why\nthis might be? Are the spheres just much larger but there are many fold less of them?\nWe thank the Reviewer for their interest in division modes of NSC driving self-renewal\nvs. differentiation. We agree that the opposite effects of long-term C1q priming on\nproliferation vs self-renewal are intriguing.\nDifferent modes of division drive self-renewal vs. differentiation in NSC - symmetric cell\ndivisions produce identical progeny, while asymmetric divisions produce two different\ntypes of progeny (PMID: 35693936). NSC division can therefore be any of the following:\nself-renewing symmetric, which produces two new NSC; self-renewing asymmetric, in\nwhich one of the two cells remains as an NSC and another initiates terminal\ndifferentiation; or self-consuming symmetric, which produces two faster proliferating\ntransit amplifying (TA) progenitors that both initiate terminal differentiation. Self-\nconsuming symmetric cell divisions leading to terminal differentiation can thus deplete\nthe originating stem cell population and result in stem cell exhaustion. Therefore, our\nobservations in which long-term C1q priming causes a proliferation rebound and\nincrease coupled with an overall decline in self-renewal could be interpreted as NSC\n20\nshifting their mode of division from self-renewing to self-consuming. This would suggest\nthat extended exposure to C1q at blood plasma concentrations could affect\nmaintenance of the NSC pool and long-term tissue turnover, providing an important\ninsight into how C1q could contribute to CNS pathological processes and impact repair\ncapacity. Critically, decline of the neural stem cell pool is posited to be a major factor in\nage-related decline of regenerative functions in the CNS, and is thought to derive from\nincreased quiescence and terminal differentiation (PMID: 31805262, PMID: 36922629).\nWe have a follow-up manuscript in preparation demonstrating importance of C1q in\nstem cell self-renewal, proliferation, and neuronal maturation in aging brain.\nWhile we alluded to this in the original manuscript, we recognize that the description\nwas quite brief. Accordingly, we have expanded our interpretation of C1q long-term\npriming effect on NSC proliferation rebound vs. decline in self-renewal in the Results to\nbetter reflect this context in Page 3, lines 9-23.\nAs per the Reviewer’s suggestion, we have also added neurosphere size to\nSupplementary Fig.1f,g. We did not detect a significant change in neurosphere size\nduring C1q [200nM]-treatment relative to non-treated controls (Response letter_Fig.6a).\nSimilarly, long-term C1q [200nM] priming caused no significant differences in\nneurosphere size relative to non-treated controls - with the exception of the observation\nthat by 6 weeks of C1q exposure no neurospheres were detected, resulting in a size\nscore of virtually 0 (Response letter_Fig.6b).\n150\n100\n50\nn\n0\n0\n2\nq\n[\nResponse letter_Fig.6. a, Neurosphere size in C1q-[200nM] treated hNSC\nrelative to non-treated controls (n = 3 independent experiments). b, C1q-\n[200nM] priming effect on neurosphere size relative to non-treated\ncontrols after medium switch and analysis under normal growth conditions\n(n = 4 independent experiments). In absence of neurospheres, the sphere\narea for analysis was scored virtually 0. Mean ± s.e.m., a 1-sample 2-\n21\n)%( \nezis \nerehpsorueN\n150\nns\nCtrl\n100\n50\n2 3 4 6\nC1q [200 nM] priming\n)%( \nezis \nerehpsorueN\nA B\nKruskal-Wallis p=0.04*\np=0.02*\np=0.02*\nns\nns Ctrl\nns\nns\nExposure to C1q (t)\ntailed t-test, b Kruskal-Wallis test with post hoc and 1-sample 2-tailed t-\ntest. ns = not significant, p > 0.05; *p ≤ 0.05. See also the Source data.\nThese data support the interpretation that there are fewer sphere initiating cells present\nover time in this clonal assay, and are generally consistent with a potential shift to self-\nconsuming symmetric divisions. A description of these added data has been included\nunder the results together with the expanded interpretation of C1q long-term priming\neffect on NSC proliferation rebound vs. decline in self-renewal (from Page 2 lines 29 to\nPage 3, line 6).\n“A reversible loss of proliferation combined with cell cycle arrest at G0 in the\nabsence of cell death (Fig.1a-j) is consistent with stem cell quiescence24,25,\nwhich would predict a decrease in self-renewal. As hypothesized, clonal\nneurosphere assays revealed a significant decrease hNSC self-renewal\nduring C1q [200nM] exposure vs. non-treated controls (Fig.1k,l). No\nsignificant change was detected in neurosphere sizes during C1q [200nM]-\ntreatment relative to non-treated controls (Supplementary Fig.1f).\nAdditionally, stem cell quiescence is distinct from cell cycle arrest during\nterminal differentiation. Analysis of total protein lysates from hNSC exposed\nto C1q [200nM] during in vitro differentiation revealed significant increases\nin undifferentiated NSC/progenitor markers, nestin and GFAPδ26, but not\nlineage commitment markers (Fig.1m). RNAseq further identified\ntranscriptomic enrichment of quiescence genes in C1q [200nM] treated\nhNSC (Fig.1n, grey). These data indicate that C1q induces quiescence in\nhNSC.\nWhile the effect of C1q on NSC proliferation was reversible after\nshort-term exposure (up to 48h, Fig.1e), CNS injury/aging can cause\nprolonged C1q elevation. We therefore tested whether long-term exposure to\nC1q has a priming effect on hNSC proliferation or self-renewal. hNSC\nneurosphere cultures were exposed to C1q for 2, 4, 6, or 8 weeks, at which\ntime C1q was withdrawn and cells returned to normal in vitro\ngrowth conditions for analysis 5 weeks later. Consistent with short-term\nexposure effects (Fig.1e), hNSC proliferation declines were reversed after\nC1q withdrawal (Fig.1o). hNSC self-renewal declines (Fig.1l), however,\npersisted after long-term priming (Fig.1p), and were exacerbated by length of\nexposure. Long-term C1q [200nM] priming caused no significant differences\nin neurosphere size relative to non-treated controls (Supplementary Fig.1g)\nsupporting the interpretation that there were fewer neurosphere initiating cells\npresent in C1q [200nM] primed group over time.”\nIn order to further clarify, we have also expanded the experimental methods and figure\nlegend, in order to make sure that the paradigm for Fig.1o,p in the revised manuscript is\nclear. For Fig.1o, hNSC were cultured as neurospheres through treatment with C1q, the\nC1q washed out, and then cells were plated in monolayer for BrdU analysis of\nproliferation. For Fig.1p, cells received the same treatment with C1q, but after\n22\ndissociation to single cells, were placed as single cells on low adherence plated to\nanalyze neurosphere initiating capacity.\nWe have also edited the Methods and Figure legends for clarity as follows:\nMethods: Proliferation, neurosphere formation, and RealTime-Glo Annexin\nV multiplex assay “For timecourse C1q priming assays, hNSC were co-\ncultured as free-floating neurospheres with or without purified C1q 48h or for\nup to 6-8 weeks followed by C1q wash out and single cell plating in\nmonolayer in GM at a density of 15,000 cells/well for BrdU or low adherence\nin GM at a density of 15 cells/well for neurosphere initiating capacity. Post-\nC1q wash BrdU incorporation time under normal growth conditions was for\n48h.”\n“Fig.1o-p Timecourse C1q-priming experiments followed by a medium\nswitch and analysis under normal growth conditions show long-term effect\nof C1q on hNSC proliferation in monolayer culture (o) and self-renewal in\nfree-floating neurospheres (p) vs. controls (n = 3 or 4 independent\nexperiments).”\n-In a number of cases, western blots are shown with bands that come from a number of\ndifferent blots including when trying to demonstrate quantitative differences (for example\nFigure 1K). In such cases, these data need to come from one blot.\nQuantitive comparisons and example western blot bands shown in this manuscript are\nfrom the same blots as shown in the Source data. Experimental conditions that are not\npart of this study have been cropped out from the example blots in Fig.1m, Fig.2b,\nFig.4y, Fig.5f, and Supplementary Fig.3a in the revised manuscript. We have revised\nthe figures to indicate this clearly by adding grey line between the lanes. Revised figure\nlegends also include this detail: “Experimental conditions that are not part of this study\nhave been cropped out from the example blots (grey line between the lanes) or\nExperimental conditions have been cropped out from the example blots for better\nvisualization (grey line between the lanes). The full original blots are in the Source\ndata.”\n-In multiple figures (for example, Figure 2c and Figure 3d) no control images are shown\nand only treatment examples are used. This makes interpretation of the data difficult. In\nthe case of Figure 2c, there is an apparent 4-fold increase in the presence of C1q which\nshould be striking in images comparing Control to 200 nM C1q.\nWe understand the reviewer’s point completely, but the limited space available makes\nthis a challenging issue to address. The intent of showing fluorescent labeling for\np53ser15 under Fig.2c, for example, was simply to show that nuclear localization was\nquantifiable, as shown in the associated graph. To make that clearer, we have revised\nthis Fig.2 to label 2c separately from the graph. Accordingly, the figure legend now\nstates:\n23\n“Fig. 2: Blood C1q concentration activates p53 signal transduction driving\ncell cycle quiescence. c, An example image of phospho-p53Ser15 nuclear\nlocalization in hNSC after ICC. d, Quantification of ICC for phospho-\np53Ser15 nuclear localization in hNSC treated with C1q [200nM] or T°Inact\nC1q [200nM] (negative control) normalized to non-treated controls (n = 3-5\nindependent experiments). Mean ± s.e.m., d Welch’s ANOVA with post hoc\ntest and 1-sample 2-tailed t-test.\nWe have revised phospho-p53Ser15 ICC figure legend also in Fig. 3:\n“Fig 3: C1q-driven p53 signal transduction is BAI1-dependent. j, Example\nimages of phospho-p53Ser15 staining in Dapi+ nucleus vs. cytoplasma with\nMitotracker+ mitochondria. k, Quantification of phospho-p53Ser15\nlocalization in nucleus in C1q-treated BAI1 WT or KO hNSC using ICC\nrelative to each control (n = 3-4 independent experiments). Mean ± s.e.m, k\n1-way ANOVA with post hoc test.\nWe would be happy to add treated and non-treated images to the extended data if this\nis judged to be a critical issue.\nIn Fig.3d PLA data, we have a similar situation. There is insufficient space to be able to\ninclude the 4 conditions quantified in panel Fig.3c. Accordingly, because the treated\ncondition has the most potential for background, we showed only treated WT versus\nKO, which we felt would provide a conservative and realistic view by which to judge the\nquantified results. Because there are no quantifiable differences detected between\ncontrol WT versus BAI1 KO, or control versus treated BAI1 KO, we feel that these\nimages are sufficient to support the quality of the staining - which was the intended goal.\nTo make that clearer, we have revised Figure 3 legend:\n“Fig.3: C1q-driven p53 signal transduction is BAI1-dependent.\nc-h, PLA analysis of protein interaction between C1q-BAI1 (c), BAI1-MDM2\n(e) or MET-MDM2 negative control (h) in C1q-treated vs. control BAI1 WT or\nKO hNSC. Example images showing PLA signal (red dots) for C1q-BAI1 (d),\nBAI1-MDM2 (f), or MET-MDM2 (g) in C1q-treated BAI1 WT vs. KO hNSC (n\n= 3-4 independent experiments). c 1-way ANOVA with post hoc test, e\nWelch’s ANOVA with post hoc test, h Kruskal-Wallis test.\nAgain, we would be happy to add treated and non-treated images to the extended data\nif this is judged to be a critical issue.\n-While the BAI1 KO and C1q dependence in this work is convincing the lack of a direct\ndemonstration of C1q and BAI1 interaction is a significant weakness. Considering that\nBAI1 recombinant protein is commercially available it should be straightforward to\ndemonstrate a direct interaction using a technique such as biolayer interferometry or\nsurface plasmon resonance or a more traditional pull-down assay. It remains possible\nthat the interaction between BAI1 and C1q is indirect.\nanalysis of the interaction kinetics between C1q and BAI1 is important. To address the\nreviewer’s immediate concerns, we performed an in situ pull-down using recombinant\nhuman C1q and BAI1 to demonstrate direct C1q-BAI1 interaction (Response\nletter_Fig.7). These data demonstrate direct interactions between C1q-BAI1, C1q-p32,\nand BAI1-p32, and that C1q-BAI1-p32 can be pulled down as a complex. The data is\nincluded in the revised manuscript as Fig.5f. We have a strong interest in studying C1q\ninteraction kinetics with all of the novel receptors we have identified (PMID: 32894219)\nusing either bio-layer interferometry (BLI) or Surface Plasmon Resonance (SPR) ;\nhowever, the expense of commercially available C1q and amount of protein\n($8000/10mg) necessary requires establishing affinity column chromatography\npurification of C1q from human plasma in our lab at UCI in order to complete those\nstudies. While in progress, we did not want to delay our resubmission further given the\nlarge amount of data we have already incorporated to the revision.\nResponse letter_Fig. 7. Direct interaction between C1q-BAI1, C1q-p32,\nand BAI1-p32 tested using in situ pull-down of human purified or\nrecombinant proteins.\n-The model with which p32 is involved with C1q and BAI1 doesn’t seem fully plausible.\nThe author’s own data suggests that p32 is in the mitochondrial matrix while C1q in\nlarge part enters the cell via endosomes. How can these two proteins specifically\ninteract when they are in different cellular compartments? This would require\ndemonstrating that both C1q are in the same place at the same time in hNSCs at high\nresolution. Moreover, if the authors propose, as in Figure 5e, that C1q enters\nendosomes and then is released from endosomes this would need to be demonstrated\nexperimentally.\nIn this manuscript we focus on whether internalized C1q interacts with intracellular p32\nin a BAI1-dependent manner. While we show that C1q is initially internalized via Rab5+\n25\nendosomes, a specific intracellular localization for C1q-p32 interaction remains to be\ndetermined. However, we thank the reviewer for this insightful question, as intracellular\nprotein interactions and trafficking are areas where much of the field of cell biology is\nseeking new understanding.\nIn endocytosis, the material to be internalized from the extracellular region is\nsurrounded by an area of plasma membrane, which then buds off inside the cell to form\na vesicle/vacuole containing the ingested material. The vesicle trafficking pathway\nincludes not only vesicle formation but also vesicle transport along the cytoskeletal\ntrack, and vesicle fusion with the target membrane. Endocytic membrane trafficking and\nsignaling are interconnected, and cargo sorting/signaling can take place at multiple\nplatforms from early/recycling endosomes to late endosomes / MVBs\n(PMID: 36129576). Endosomes communicate with other organelles through membrane\ncontact sites (MCS). Most notably, tubules emanating from the endoplasmic reticulum\n(ER) contact endosomes within a distance of 30 nm or shorter (PMID: 32258039). In\nthis, the ER is a dynamic organelle that performs many functions including, lipid\nmetabolism, calcium storage, and protein folding - and also makes contact sites with\nmultiple membranes including mitochondria (PMID: 26041457).\nSo far, we know that p32 protein is translated in the ER as a precursor (pre-p32)\ncontaining a mitochondrial targeting sequence which is proteolytically removed\n(PMID: 9305894, PMID: 9531316). Even though, p32 is mainly localized in the\nmitochondria, it has been also detected in the ER (PMID: 11493647), nucleus\n(PMID: 11243856), and cell surface. Due to the localization of p32 in these different\nsubcellular compartments, and the capacity of p32 to interact with several proteins, p32\nis thought to function as a chaperone protein (https://www.uniprot.org/uniprotkb/\nQ07021/entry#interaction). In agreement with this interpretation, we report detection of\np32 in both mitochondrial and insoluble/particulate fraction of subcellular structure\nbound proteins but not at the cell surface (Fig.4r,s).\nIn hNSC, internalized C1q is detected at least in endosomes and endosomal\nmultivesicular bodies (Fig.4f,h). We completed unbiased nanoLC-MS/MS of pull-down\nexperiments in hNSC using internalized C1q as bait, seeking to identify intracellular\ninteracting proteins (prey). We then conducted subcellular enrichment analysis of prey\nproteins that were significantly different in C1q vs untreated samples, identifying several\nspecific compartments involved in receptor-mediated endocytic trafficking of C1q (Fig.\n4i, Supplementary Fig.4a). These data suggest that internalized C1q is present in\nseveral locations permitting interaction with p32. However, there are many additional\nquestions for future studies, including identification of target membranes, tethering\nprotein/protein complexes coordinating C1q cargo, specific locations of C1q-p32\ninteraction, and the biological processes regulated by these interactions.\nMinor:\n-Page 2 Line 1 BCB not defined. Perhaps BSB typo?\n(BCB) to blood-brain/spinal cord-barrier (BBB/BSB) in page 2 line 2, and check the rest\nof the manuscript for consistency regarding the terminology.\n-In a number of cases “Blood-derived C1q concentrations” is used. This is confusing as\nthe data shown here actually has nothing to do with “Blood-derived C1q concentration\npromotes.” Consider modifying this terminology for clarity.\nPer Reviewer’s request, we have changed the original subtitle “Blood-derived C1q\npromotes hNSC quiescence” to “C1q promotes hNSC quiescence” page 2 line 11.\n-In Figure 3B, this is the only blot shown as white bands on black background when all\nof the rest of the blots are shown inverted. This is confusing and can lead to confusion\nover whether these data are western or an agarose gel.\nAgreed; We have changed Fig. 3b to match the style of rest of the western blots.\n-In Figure 4i, pull down based proteomics data is introduced but these data are hardly\ndescribed or mentioned at all and only shown as a cartoon. If these data are significant\nenough to be shown in the main figures they should be described and interpreted in a\nmore fulsome manner.\nWe thank the Reviewer for bringing this to our attention - we recognize that the\ndescription of Fig.4i was quite brief. To address this comment, we have changed the\nfigure format into a bubble plot for clarity and expanded the text accordingly. Briefly,\nthese data represent unbiased nanoLC-MS/MS of pull-down experiments in hNSC using\ninternalized C1q as bait, seeking to identify intracellular interacting proteins (prey). We\nthen conducted subcellular enrichment analysis of prey proteins that were significantly\ndifferent in C1q vs. untreated samples, identifying several specific compartments\ninvolved in receptor-mediated endocytic trafficking of C1q (Fig.4i, Supplementary Fig.\n4a; Response letter_Fig.8). The complete list of proteins that were significantly different\nin C1q vs. untreated samples, and the subcellular compartments identified using\nsubcellular enrichment analysis, is now shown as Supplementary Fig.4a and is also\nincluded in the source data.\nthat were significantly different in C1q vs. untreated samples in several specific\ncompartments involved in receptor-mediated endocytic trafficking of C1q in hNSC after\nunbiased nanoLC-MS/MS analysis. A FDR-adjusted p-value threshold of ≤0.05 was\nconsidered significant (n = 4 or 8 independent experiments).\nIn order to further clarify, we have also expanded the figure legend as shown in\nResponse letter_Fig 8., and experimental methods, in order to make sure that the\nparadigm for Fig.4i in the revised manuscript is clear.\nMethods, Page 17: “Cross-linking, co-immunoprecipitation, and LC-MS/\nMS\n500µg of C1q was reversibly biotinylated with an Ez-link NHS-SS-\nBiotinylation kit (ThermoScientific, PI21441) following the manufacturer\ninstructions. Briefly, to prepare biotin-labeled C1q, 1mg purified human\nC1q was resuspended in 1mL Milli-Q H2O (1mg/mL) and half of the vial\n(500µg of C1q) was buffer exchanged to PBS using Zeba spin desalting\ncolumn 7K MWCO (ThermoFisher Scientific) was following the\nmanufacturer instructions. C1q in PBS (1.25nM C1q) was incubated with\na 20-fold molar excess (25nM) of NHS-SS-Biotin for 1h RT in agitation.\nThe non-bound biotin was removed by buffer exchange using a Zeba spin\ndesalting column 7K MWCO (500µl final volume). 10µl aliquot (1:30\ndilution) was used to determine C1q protein concentration by BCA\nanalysis. A total of 60-80% of the original amount of C1q was recovered\nafter the biotinylation process. An aliquot of the biotinylated C1q was\nanalyzed by Western blot to identify biotinylated C1q.\nhNSC were grown as a monolayer at PLO-laminin-coated 6-well\nplates. Prior to the experiment, the cells were washed three times with\nPBS (pH 7.2) and 200nM of biotinylated C1q (bait) or an equal volume of\nGM (non-treated controls) was added to the cells for 15min or 30min at\nRT. Analysis was performed as four independent experiments with one\nsample well per each. A cell membrane permeable DSP with 12Å spacer\narm (ThermoScientific, PI22586) was used for intracellular crosslinking of\nthe treated samples according to the manufacturer instructions. Briefly,\nthe cells were washed three times with PBS (pH 7.2) and exposed to\n2mM of DSP at RT for 30min. The sample was quenched with three 5min\nwashes of 25mM Tris in PBS. The cells were lysed using Pierce IP lysis\nbuffer (ThermoScientific, PI87787) supplemented with protease and\nphosphatase inhibitors cocktail on ice. Lysates were centrifuged 13,000g\nfor 10min at 4°C and for pull-down supernatants were incubated on\nwashed Pierce Streptavidin Magnetic beads (ThermoScientific,\nPI188816) for 1h at RT followed by washes to remove non-specific\nbinding partners. For proteolytic protein digestion, magnetic beads with\nprotein were washed with HPLC grade H2O. 2M urea with 25mM\n(NH4)HCO3 was added into slurry in 1:1 ratio followed by 15min\nincubation with DTT as a final concentration of 2mM at 37°C. Next, the\n28\nslurry was incubated with iodoacetamide at final concentration of 4mM for\n30min protected from light followed by 15min quenching with DTT as a\nfinal concentration of 2mM at 37°C. 25 mM (NH4)HCO3 was added into\nthe slurry in 1:1 ratio to dilute urea into final 1M followed by overnight\nincubation with trypsin at a final concentration of 20ng/μL at 37°C.\nDigestion was stopped by 0.1% formic acid and the protein solution was\ndesalted using C18 column (Agilent) according to the manufacturer\ninstructions.\nPeptide digests were subjected to LC MS/MS analysis using an\nUltiMate 3000 RSLC system (Thermo Fisher Scientific) coupled in-line to\nan Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific).\nReverse-phase separation was performed on a 50 cm x 75 μm I.D.\nAcclaim® PepMap RSLC column. Peptides were eluted using a gradient\nof 4% to 22% B over 70min at a flow rate of 300nL/min (solvent A: 100%\nH2O, 0.1% formic acid; solvent B: 100% acetonitrile, 0.1% formic acid).\nEach cycle consisted of one full Fourier transform scan mass spectrum\n(375–1500 m/z, resolution of 120,000 at m/z 400) followed by data-\ndependent MS/MS scans acquired in the linear ion with HCD at NCE\n30% trap at top speed for 3s. Target ions already selected for MS/MS\nwere dynamically excluded for 30s. Protein identification and label-free\nquantitation were carried out using MaxQuant as described109. Raw\nspectrometric files were searched using MaxQuant version 1.6.0.16\nagainst a FASTA of the complete human proteome obtained from\nSwissProt Feb 2020 version. The first search peptide tolerance was set to\n20 ppm, with main search peptide tolerance set to 4.5 ppm. Trypsin was\nset as the digestive enzyme with max 2 missed cleavages. Methione\noxidation and protein N-terminal acetylation were wet as variable\nmodifications, while cysteine carbamidomethylation was set as a fixed\nmodification. Peptide spectra match and protein FDRs were both set as\n0.01. The generated raw mass spectrometry data was preprocessed and\nanalyzed by Differential Enrichment Pipeline package (DEP) version 1.22\n(Bioconductor)110. For quality control included filtering proteins with high\nmissing values, assessing reproducibility, and addressing batch effect,\nthe tool’s default settings were applied. Quantitative values were\nnormalized using variance stabilizing transformation (VST) to minimize\ntechnical variation and improve comparability across samples. For\ndifferential expression analysis, the samples treated with biotinylated C1q\nper each independent experiment were pooled together and protein-wise\nlinear models combined with empirical Bayesian statistics, as\nimplemented in the DEP package, were used to identify differentially\nexpressed proteins between two conditions bait versus non-treated\ncontrol. Adjusted p-values were calculated using the Benjamini-Hochberg\nmethod, with proteins showing an FDR-adjusted p-value ≤0.05\nconsidered significantly differentially expressed. The significant proteins\nare listed in the Source data. Hypergeometric tests in SubCellViz R\npackage111 was employed to analyze the subcellular localization of the\n29\ndifferentially expressed proteins to illustrate protein distribution and\nidentify compartment-specific functional patterns. Protein identifiers were\nannotated to subcellular compartments using Gene Ontology Cellular\nComponent (GO-CC) and UniProt databases. Enrichment analysis was\nperformed using hypergeometric tests, and compartments with FDR-\nadjusted p-values ≤ 0.05 were identified as significantly enriched. All\nanalyses were performed by investigators blinded to experimental\nconditions/groups.”\nReviewer #4 (Remarks to the Author):\n\"I co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\"\nThank you, we appreciate the Reviewer’s time and effort to improve our manuscript.\nWe appreciate the valuable feedback on our manuscript. We have carefully considered\neach comment and made revisions accordingly. Additionally, as requested, all of the\nfigures have been formatted to match Nature Communications requirements, and the\nsource data files with raw data have been included in this resubmission. Below,\nwe address each of the points raised.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis manuscript reports effects of C1q on human neural stem cells (hNSCs) mediated\nby the receptor BAI1. The authors describe effects of C1q on hNSC proliferation and\nother aspects of the cells’ physiology. In parallel studies, the authors provide insight into\nthe signaling pathways that are engaged when C1q binds BAI1, specifically i) regulation\nof mdm2 to control p53 and ii) endocytosis of C1q to modulate p32. These findings are\nnovel and interesting.\nThe authors were very responsive to the reviewers' comments and have addressed all\nof the reviewers' concerns following the initial review. The textual changes and new data\nadded by the authors have significantly strengthened the manuscript.\nWe thank Reviewer for their time and effort invested to improve our manuscript.\nReviewer #2 (Remarks to the Author):\nThe authors have addressed to the largest extend the queries and concerns raised by\nthis reviewer satisfactorily.\nThe only 'negative' remaining would be that the authors have missed an opportunity\npinpoint then intracellular location of the C1q/p32 location better - this was also raised\nby reviewer 3. I am not sure why as this would have elevated to the work substantially\nand performing higher resolution/quality staining of mitochondria as suggested by this\nreviewer (or endosomal compartments as indicated by the authors) is not difficult.\nHowever, this reviewer does do not feel sufficiently strongly about this matter to ask for\nanother revision.\nWe thank Reviewer for the insightful questions, and the time and effort invested to\nimprove our manuscript. Based on our current knowledge of the possible intracellular\nlocalization of p32 and the several specific intracellular compartments involved in\nreceptor-mediated endocytic trafficking of C1q intracellular we hypothesize that the\nlocation of C1q-p32 intracellular interactions is far more complex than anticipated (see\nC1q-p32 PLA interaction puncta in Fig.4V). Therefore we would prefer to address this\nquestion in a follow-up manuscript in preparation reporting additional novel biological\nprocesses regulated by intracellular C1q.\nReviewer #3 (Remarks to the Author):\nThe authors have suitably addressed a number of my concerns and the manuscript has\nimproved substantially. However, a couple of points remain:\n1) I congratulate the authors for carrying out the pull down experiment using each of the\ninteracting proteins. These data are now in Figure 5F. The manner, however, in which\nthese data are presented makes it nearly impossible to interpret. For example, it\nappears that BAI1 is used as the bait in all lanes (except 3 and 4) shown in these blots\nand the probed with antibodies above? The presentation of these data could be\nsignificantly improved by indicating bait and prey in each case. It took this reviewer, a\nsignificant amount of time reviewing the methods and looking at the figure and legend to\nget an idea of what is going in with that data. The data should be interpretable by simply\nlooking at the figure and legend. In addition, in lanes 3 and 4, it appears that no BAI1\nbait was used and yet C1q and p32 are pulled down. How can that be if only BAI1 has\nthe his-tag to bind to the magnetic beads? Is this result of non-specific binding of C1q/\np32 to the beads or something else? If the former, this would seriously impact these\ndata and call them into question.\nWe thank Reviewer for their extra time and attention to detail to improve the readability\nof our manuscript. We utilized His-tagged BAI1 as the bait for all pulldowns except the\nC1q-p32 pulldown. To test C1q-p32 interactions, we used His-tagged p32 as the bait\ninstead of the untagged p32 utilized in the other pulldowns. We apologize for failing to\ninclude the details of which His-tagged bait protein was used in each lane; we have\nupdated the figure and figure legend as shown below, which we believe addresses this\nFigure 5\nissue. We hope that these changes have improved the interpretability of our pulldown\nresults (Fig. 5f). B C\n1.5\n1\n0.5\n0\n-0.5 140\n-1\n-1.5\n120\n100\n80\n60\n40\n20\n0\nml ml ml n e n e ct]\n2\n0 n g\n5\n/\n0\n0 n g/\n8\n0\n+\nμ g\nR\n/ ot e n o\nR\not e\nm\nn o\nl [\nT °i n a\nml μ\ng/\ng/ 0\nμ 8\n0 8\nL\nMitotracker / Dapi Mito segmentation / Dapi\nWT KO WT KO\nCtrl [GM] Ctrl [GM] Ctrl [GM] Ctrl [GM] N\nWT KO WT KO\nC1q [200nM] C1q [200nM] C1q [200nM] C1q [200nM] 3 µm\n)%\n(\ng\nA D\n[C1q] [C1q]\nCtrl 1-way ANOVA p<0.0001****\nC1q 200 nM\nns I J\np=0.003**\np<0.0001****\np<0.0001****\np<0.0001****\nCtrl p<0.0001****\np<0.0001**** ns\np<0.0001************\nH ****\np=0.004**\nns ns\nCtrl\n****\n200nM\nMMMM M M\n[C1q] n n nmm n\nB O o X u P n H d O S Free N $N A AD D GH$ H LYCOLYS F I r S ee 0. 1 1\n+\n. R 0 o 2 t e 0 n R o 0 n o e t e 1\nT\nn 0 o ̊I n n e a 1 c 0 t G 2 0 0\nM\nn\n0\n0 2\n120\n100\n80\n60\n40\n20\n0\n[C1q] 0 0\n8 8\nq q 1 1\nC C\nT O W K 4 4\n2 2\nFig.5: C1q-p32 intracellular interaction drives p32 instability and aerobic glycolysis in BAI1-dependent manner.\na-b, Imagestream analysis shows BAI1 KO decreases the proportion of in-focus single hNSC with intracellular\nC1q-p32 co-localization (a) and the overall quantity of intracellular C1q-p32 co-localization signal per hNSC\nwithin this sub-population (b) relative to C1q [200 nM]-treated BAI1 WT (ctrl, dashed line) (n = 3 independent\nexperiments). c, Example images showing PLA signal (red dots) for C1q-p32 in C1q-treated BAI1 WT vs. KO\nhNSC. d, PLA analysis of intracellular C1q-p32 interaction in C1q-treated BAI1 WT or KO hNSC vs. controls\n(n = 4-5 independent experiments). e, Western blot analysis of total p32 in C1q-treated vs. non-treated BAI1\nWT or KO hNSC (n = 3 independent experiments). Optical band densities normalized to βActin. f, His-tag pull-\ndown of purified human proteins in situ demonstrates direct interaction between C1q-BAI1, C1q-p32, and\nBAI1-p32. Bait red, Western blot antibodies blue. Blots have been cropped and stitched together for better\nvisualization (grey line between the lanes). The full original blots are in the Source data. g, Schematic of the\nhypothesized metabolic outcome for BAI1-dependent intracellular C1q-p32 interaction and decreased p32\nstability. Created with BioRender.com. h-i, C1q [200nM] effect on transcriptional activation of genes\nassociated with glycolysis from RNA-seq (n = 3 independent experiments). A graphical view of the enrichment\nscore for the glycolysis gene set (h), and heat map of abundance of the genes (i). Positive scores (red)\nsignificant enrichment (FDR adjusted p-value ≤0.05) in h. Red colors up-regulation and blue colors down-\nregulation in i. j, Example fluorescence life-time imaging microscopy (FLIM) images showing bound vs. free\nNADH/NAD in control and C1q [200nM]-treated hNSC. k, FLIM analysis of NADH/NAD+ redox ratio\ncorresponding to glycolytic and OXPHOS states in C1q vs. Rotenone-treated or control hNSC (n = 5-9 cells\nper group per timepoint). I, Example images of mitochondria morphology based on Mitotracker staining and\nsegmentation in non-treated and C1q-treated BAI1 WT and KO hNSC. m, Mitochondria morphology analysis\nin C1q-treated BAI1 WT or KO hNSC relative to each control (n = 4-5 independent experiments). n-o, Basal\noxygen consumption rate (OCR) (n) and extracellular acidification rate (ECAR ) (o) in C1q-treated BAI1 WT or\nKO hNSC relative to each control (n = 3-4 independent experiments). Mean ± s.e.m., a,b 1-sample 2-tailed t-\ntest, d,n unpaired 1-tailed Welch’s t-test with 1-sample 2-tailed t-test, e,o unpaired 1-tailed t-test and 1-sample\n2-tailed t-test, m unpaired 1-tailed t-test or unpaired 1-tailed Welch’s t-test with 1-sample 2-tailed t-test, k 1-\nway ANOVA with post hoc test and 1-sample 2-tailed t-test. ns = not significant, p>0.05; *p≤0.05; **p≤0.01;\n***p≤0.001; ****p≤0.0001. See also Supplementary Figs. 6 and 7, and Source data.\n#\nkrowten\negraL\n120\n100\n80\n60\nM] M]\nn n\nW T [ 2 0 0 O [ 2 0 0\nK\np=0.03*\nns\nCtrl\n*\nT O[C1q] W K\n23p\nE\n* p=0.02\nns\nCtrl\n*\n200 nM Control T O T O W K W K\n33 kDa p32\n42 kDa βActin\nK\nM\n300\n250\n200\n150\n100\n50\n0\n[C1q] 0 0\n8 8\nq q 1 1\nC C\nT O W K 4 4\n2 2\n#\neatcnuP\np=0.04*\nns\nCtrl\n*\nT O W K\nO\n150\n100\n50\nW K\n)%\n(\nRCO\np=0.05*\nns 250\n150\n**\n100\n50\n0\n[C1q] T O\nW K\n)%\n(\nRACE\n120\n100\n80\n60\n40\n20\np=0.003**\n*\nns\nCtrl\nnoitazilacoloc\n23p-q1C\nllec\nrep\n100\n80\n60 *\n40\n20\nnoitazilacoloc\n23p-q1C\n%\nsllec\ndetag\nrep\nF PLA C1q-p32\nWT\n3 µm\nWT\nC1q [200nM]\n*\nKO\nC1q [200nM]\n300\n250\n160\n120\n80\n40\n0\n0 0 8 8\nq q\n1 1 C C\nT O\nW K 4 4 2 2\n#\nkrowten\nllamS\n4000\n3000\n2000\n1000\n600\n500\n400\n300\n200\n100\np=0.07\nns\nCtrl\n*\nT O[C1q] W K\n23p-q1C\nWB: Antibodies\nG0 B AI 1 C 1 q fu p l3l 2fig C u1 qre g B ArIo 1 u p p3 2ed B AI 1 C 1 q p 3 2\nG0 kDa\n𝜶 𝜶 𝜶 𝜶 𝜶 𝜶 𝜶 𝜶 𝜶\nG2 M G2 M p=0.03*\nG1 *\nS G1 S\n110\n*\nHis-BAI1 His-p32 His-BAI1 His-BAI1 Bait\nG\nC1q C1q BAI1 BAI1\nC1q full figure with text separate\nBAI1 p32 p32\np32 p32\nBAI1 p32 p32\np32 p32 C1q p32 p32\np32\nAerobic OXPHOS\nGlycolysis\nResponse letter_Fig. 1 His-tag pull-down of purified human proteins in situ\ndemonstrates direct interaction between C1q-BAI1, C1q-p32, and BAI1-p32.\nBait red, Western blot antibodies blue. Blots have been cropped and\nstitched together for better visualization (grey line between the lanes). The\nfull original blots are in the Source data.\nIn addition, we have included the details in the Methods section, page 19, row 11 as\nfollows:\n“Pull-down protein interaction analysis\nDirect interaction between C1q, BAI1 and p32 in situ was tested by\npulldown assays using purified human proteins following the manufacturer\ninstructions. Briefly, 5 µg His-tag recombinant human BAI1 (SinoBiological,\n4969-BA) or His-tag p32 (SinoBiological, 11874-H08E) were prepared as\nbait in a 1x binding/wash buffer (50 mM sodium phosphate, 300 mM NaCl,\n0.01% Tween-20) and immobilized on 2 mg magnetic beads (Dynabeads\nHis-Tag Isolation and Pulldown, Invitrogen, 10103D) by incubation on a\nrocker for 10 min at RT. Tubes containing the bait protein immobilized on\nbeads were placed on a magnet (Invitrogen, 12321D) and the supernatant\ncontaining excess bait protein was collected for Western blot validation.\nBeads were washed four times with 1x binding/wash buffer to remove any\nremaining excess bait protein. 5 µg of prey proteins purified human C1q\n(MyBioSource, MBS147305) and recombinant untagged human p32 (LSBio,\nLS-G3375-20) were prepared in a 1x pulldown buffer (3.25 mM sodium\nphosphate, 70 mM NaCl, 0.01% Tween-20) and incubated with the\nimmobilized bait protein on a rocker for 15 min at RT. Prior to pulldown\nprotein capture, tubes containing the immobilized bait-prey protein\ncomplexes were placed on a magnet and washed four times with 1x\nbinding/wash buffer to remove any remaining unbound prey proteins. Bait\nprotein and bound prey proteins were eluted using his-elution buffer (300\nmM imidazole, 50 mM sodium phosphate, 300 mM NaCl, 0.005%\nTween-20) and the eluted samples were analyzed using Western\nblotting. The original blots are in the Source data.”\n2) Related to the above, the authors indicate in a number of instances in the response\nthat \"The full original blots are in the Source data.\" This reviewer looked through every\nfile in the manuscript tracking system and could not locate any original blots. These\nblots would help to interpret the data mentioned in point #1 above.\nWe apologize for the confusion. We were not able to upload the source file (an excel\nfile) directly due to its complexity and size into the Nature Communications portal (only\npdf allowed). The document was uploaded for us by the submission support and we\nwere not able to validate the document afterwards. Our best guess is that something\nhappened and the photos of the western blot membranes for Fig. 1m, Fig. 2b, Fig. 3b,\nFig. 3i, Fig. 4r, Fig. 4s, Fig.4y, Fig. 5e, Fig. 5f, and Supplementary Fig. 3a that we had\nattached into the corresponding sheets were lost during this process. The source data\nfile with the photos of the western blot membranes is included into this resubmission\nagain.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nWe thank Reviewer for their time co-reviewing our manuscript.\nWe appreciate the valuable feedback on our manuscript. We have carefully considered\neach comment and made revisions accordingly. Additionally, as requested, all of the\nfigures have been formatted to match Nature Communications requirements, and the\nsource data files with raw data have been included in this resubmission. Below,\nwe address each of the points raised.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis manuscript reports effects of C1q on human neural stem cells (hNSCs) mediated\nby the receptor BAI1. The authors describe effects of C1q on hNSC proliferation and\nother aspects of the cells’ physiology. In parallel studies, the authors provide insight into\nthe signaling pathways that are engaged when C1q binds BAI1, specifically i) regulation\nof mdm2 to control p53 and ii) endocytosis of C1q to modulate p32. These findings are\nnovel and interesting.\nThe authors were very responsive to the reviewers' comments and have addressed all\nof the reviewers' concerns following the initial review. The textual changes and new data\nadded by the authors have significantly strengthened the manuscript.\nWe thank Reviewer for their time and effort invested to improve our manuscript.\nReviewer #2 (Remarks to the Author):\nThe authors have addressed to the largest extend the queries and concerns raised by\nthis reviewer satisfactorily.\nThe only 'negative' remaining would be that the authors have missed an opportunity\npinpoint then intracellular location of the C1q/p32 location better - this was also raised\nby reviewer 3. I am not sure why as this would have elevated to the work substantially\nand performing higher resolution/quality staining of mitochondria as suggested by this\nreviewer (or endosomal compartments as indicated by the authors) is not difficult.\nHowever, this reviewer does do not feel sufficiently strongly about this matter to ask for\nanother revision.\nWe thank Reviewer for the insightful questions, and the time and effort invested to\nimprove our manuscript. Based on our current knowledge of the possible intracellular\nlocalization of p32 and the several specific intracellular compartments involved in\nreceptor-mediated endocytic trafficking of C1q intracellular we hypothesize that the\nlocation of C1q-p32 intracellular interactions is far more complex than anticipated (see\nC1q-p32 PLA interaction puncta in Fig.4V). Therefore we would prefer to address this\nquestion in a follow-up manuscript in preparation reporting additional novel biological\nprocesses regulated by intracellular C1q.\nReviewer #3 (Remarks to the Author):\nThe authors have suitably addressed a number of my concerns and the manuscript has\nimproved substantially. However, a couple of points remain:\n1) I congratulate the authors for carrying out the pull down experiment using each of the\ninteracting proteins. These data are now in Figure 5F. The manner, however, in which\nthese data are presented makes it nearly impossible to interpret. For example, it\nappears that BAI1 is used as the bait in all lanes (except 3 and 4) shown in these blots\nand the probed with antibodies above? The presentation of these data could be\nsignificantly improved by indicating bait and prey in each case. It took this reviewer, a\nsignificant amount of time reviewing the methods and looking at the figure and legend to\nget an idea of what is going in with that data. The data should be interpretable by simply\nlooking at the figure and legend. In addition, in lanes 3 and 4, it appears that no BAI1\nbait was used and yet C1q and p32 are pulled down. How can that be if only BAI1 has\nthe his-tag to bind to the magnetic beads? Is this result of non-specific binding of C1q/\np32 to the beads or something else? If the former, this would seriously impact these\ndata and call them into question.\nWe thank Reviewer for their extra time and attention to detail to improve the readability\nof our manuscript. We utilized His-tagged BAI1 as the bait for all pulldowns except the\nC1q-p32 pulldown. To test C1q-p32 interactions, we used His-tagged p32 as the bait\ninstead of the untagged p32 utilized in the other pulldowns. We apologize for failing to\ninclude the details of which His-tagged bait protein was used in each lane; we have\nupdated the figure and figure legend as shown below, which we believe addresses this\nFigure 5\nissue. We hope that these changes have improved the interpretability of our pulldown\nresults (Fig. 5f). B C\n1.5\n1\n0.5\n0\n-0.5 140\n-1\n-1.5\n120\n100\n80\n60\n40\n20\n0\nml ml ml n e n e ct]\n2\n0 n g\n5\n/\n0\n0 n g/\n8\n0\n+\nμ g\nR\n/ ot e n o\nR\not e\nm\nn o\nl [\nT °i n a\nml μ\ng/\ng/ 0\nμ 8\n0 8\nL\nMitotracker / Dapi Mito segmentation / Dapi\nWT KO WT KO\nCtrl [GM] Ctrl [GM] Ctrl [GM] Ctrl [GM] N\nWT KO WT KO\nC1q [200nM] C1q [200nM] C1q [200nM] C1q [200nM] 3 µm\n)%\n(\ng\nA D\n[C1q] [C1q]\nCtrl 1-way ANOVA p<0.0001****\nC1q 200 nM\nns I J\np=0.003**\np<0.0001****\np<0.0001****\np<0.0001****\nCtrl p<0.0001****\np<0.0001**** ns\np<0.0001************\nH ****\np=0.004**\nns ns\nCtrl\n****\n200nM\nMMMM M M\n[C1q] n n nmm n\nB O o X u P n H d O S Free N $N A AD D GH$ H LYCOLYS F I r S ee 0. 1 1\n+\n. R 0 o 2 t e 0 n R o 0 n o e t e 1\nT\nn 0 o ̊I n n e a 1 c 0 t G 2 0 0\nM\nn\n0\n0 2\n120\n100\n80\n60\n40\n20\n0\n[C1q] 0 0\n8 8\nq q 1 1\nC C\nT O W K 4 4\n2 2\nFig.5: C1q-p32 intracellular interaction drives p32 instability and aerobic glycolysis in BAI1-dependent manner.\na-b, Imagestream analysis shows BAI1 KO decreases the proportion of in-focus single hNSC with intracellular\nC1q-p32 co-localization (a) and the overall quantity of intracellular C1q-p32 co-localization signal per hNSC\nwithin this sub-population (b) relative to C1q [200 nM]-treated BAI1 WT (ctrl, dashed line) (n = 3 independent\nexperiments). c, Example images showing PLA signal (red dots) for C1q-p32 in C1q-treated BAI1 WT vs. KO\nhNSC. d, PLA analysis of intracellular C1q-p32 interaction in C1q-treated BAI1 WT or KO hNSC vs. controls\n(n = 4-5 independent experiments). e, Western blot analysis of total p32 in C1q-treated vs. non-treated BAI1\nWT or KO hNSC (n = 3 independent experiments). Optical band densities normalized to βActin. f, His-tag pull-\ndown of purified human proteins in situ demonstrates direct interaction between C1q-BAI1, C1q-p32, and\nBAI1-p32. Bait red, Western blot antibodies blue. Blots have been cropped and stitched together for better\nvisualization (grey line between the lanes). The full original blots are in the Source data. g, Schematic of the\nhypothesized metabolic outcome for BAI1-dependent intracellular C1q-p32 interaction and decreased p32\nstability. Created with BioRender.com. h-i, C1q [200nM] effect on transcriptional activation of genes\nassociated with glycolysis from RNA-seq (n = 3 independent experiments). A graphical view of the enrichment\nscore for the glycolysis gene set (h), and heat map of abundance of the genes (i). Positive scores (red)\nsignificant enrichment (FDR adjusted p-value ≤0.05) in h. Red colors up-regulation and blue colors down-\nregulation in i. j, Example fluorescence life-time imaging microscopy (FLIM) images showing bound vs. free\nNADH/NAD in control and C1q [200nM]-treated hNSC. k, FLIM analysis of NADH/NAD+ redox ratio\ncorresponding to glycolytic and OXPHOS states in C1q vs. Rotenone-treated or control hNSC (n = 5-9 cells\nper group per timepoint). I, Example images of mitochondria morphology based on Mitotracker staining and\nsegmentation in non-treated and C1q-treated BAI1 WT and KO hNSC. m, Mitochondria morphology analysis\nin C1q-treated BAI1 WT or KO hNSC relative to each control (n = 4-5 independent experiments). n-o, Basal\noxygen consumption rate (OCR) (n) and extracellular acidification rate (ECAR ) (o) in C1q-treated BAI1 WT or\nKO hNSC relative to each control (n = 3-4 independent experiments). Mean ± s.e.m., a,b 1-sample 2-tailed t-\ntest, d,n unpaired 1-tailed Welch’s t-test with 1-sample 2-tailed t-test, e,o unpaired 1-tailed t-test and 1-sample\n2-tailed t-test, m unpaired 1-tailed t-test or unpaired 1-tailed Welch’s t-test with 1-sample 2-tailed t-test, k 1-\nway ANOVA with post hoc test and 1-sample 2-tailed t-test. ns = not significant, p>0.05; *p≤0.05; **p≤0.01;\n***p≤0.001; ****p≤0.0001. See also Supplementary Figs. 6 and 7, and Source data.\n#\nkrowten\negraL\n120\n100\n80\n60\nM] M]\nn n\nW T [ 2 0 0 O [ 2 0 0\nK\np=0.03*\nns\nCtrl\n*\nT O[C1q] W K\n23p\nE\n* p=0.02\nns\nCtrl\n*\n200 nM Control T O T O W K W K\n33 kDa p32\n42 kDa βActin\nK\nM\n300\n250\n200\n150\n100\n50\n0\n[C1q] 0 0\n8 8\nq q 1 1\nC C\nT O W K 4 4\n2 2\n#\neatcnuP\np=0.04*\nns\nCtrl\n*\nT O W K\nO\n150\n100\n50\nW K\n)%\n(\nRCO\np=0.05*\nns 250\n150\n**\n100\n50\n0\n[C1q] T O\nW K\n)%\n(\nRACE\n120\n100\n80\n60\n40\n20\np=0.003**\n*\nns\nCtrl\nnoitazilacoloc\n23p-q1C\nllec\nrep\n100\n80\n60 *\n40\n20\nnoitazilacoloc\n23p-q1C\n%\nsllec\ndetag\nrep\nF PLA C1q-p32\nWT\n3 µm\nWT\nC1q [200nM]\n*\nKO\nC1q [200nM]\n300\n250\n160\n120\n80\n40\n0\n0 0 8 8\nq q\n1 1 C C\nT O\nW K 4 4 2 2\n#\nkrowten\nllamS\n4000\n3000\n2000\n1000\n600\n500\n400\n300\n200\n100\np=0.07\nns\nCtrl\n*\nT O[C1q] W K\n23p-q1C\nWB: Antibodies\nG0 B AI 1 C 1 q fu p l3l 2fig C u1 qre g B ArIo 1 u p p3 2ed B AI 1 C 1 q p 3 2\nG0 kDa\n𝜶 𝜶 𝜶 𝜶 𝜶 𝜶 𝜶 𝜶 𝜶\nG2 M G2 M p=0.03*\nG1 *\nS G1 S\n110\n*\nHis-BAI1 His-p32 His-BAI1 His-BAI1 Bait\nG\nC1q C1q BAI1 BAI1\nC1q full figure with text separate\nBAI1 p32 p32\np32 p32\nBAI1 p32 p32\np32 p32 C1q p32 p32\np32\nAerobic OXPHOS\nGlycolysis\nResponse letter_Fig. 1 His-tag pull-down of purified human proteins in situ\ndemonstrates direct interaction between C1q-BAI1, C1q-p32, and BAI1-p32.\nBait red, Western blot antibodies blue. Blots have been cropped and\nstitched together for better visualization (grey line between the lanes). The\nfull original blots are in the Source data.\nIn addition, we have included the details in the Methods section, page 19, row 11 as\nfollows:\n“Pull-down protein interaction analysis\nDirect interaction between C1q, BAI1 and p32 in situ was tested by\npulldown assays using purified human proteins following the manufacturer\ninstructions. Briefly, 5 µg His-tag recombinant human BAI1 (SinoBiological,\n4969-BA) or His-tag p32 (SinoBiological, 11874-H08E) were prepared as\nbait in a 1x binding/wash buffer (50 mM sodium phosphate, 300 mM NaCl,\n0.01% Tween-20) and immobilized on 2 mg magnetic beads (Dynabeads\nHis-Tag Isolation and Pulldown, Invitrogen, 10103D) by incubation on a\nrocker for 10 min at RT. Tubes containing the bait protein immobilized on\nbeads were placed on a magnet (Invitrogen, 12321D) and the supernatant\ncontaining excess bait protein was collected for Western blot validation.\nBeads were washed four times with 1x binding/wash buffer to remove any\nremaining excess bait protein. 5 µg of prey proteins purified human C1q\n(MyBioSource, MBS147305) and recombinant untagged human p32 (LSBio,\nLS-G3375-20) were prepared in a 1x pulldown buffer (3.25 mM sodium\nphosphate, 70 mM NaCl, 0.01% Tween-20) and incubated with the\nimmobilized bait protein on a rocker for 15 min at RT. Prior to pulldown\nprotein capture, tubes containing the immobilized bait-prey protein\ncomplexes were placed on a magnet and washed four times with 1x\nbinding/wash buffer to remove any remaining unbound prey proteins. Bait\nprotein and bound prey proteins were eluted using his-elution buffer (300\nmM imidazole, 50 mM sodium phosphate, 300 mM NaCl, 0.005%\nTween-20) and the eluted samples were analyzed using Western\nblotting. The original blots are in the Source data.”\n2) Related to the above, the authors indicate in a number of instances in the response\nthat \"The full original blots are in the Source data.\" This reviewer looked through every\nfile in the manuscript tracking system and could not locate any original blots. These\nblots would help to interpret the data mentioned in point #1 above.\nWe apologize for the confusion. We were not able to upload the source file (an excel\nfile) directly due to its complexity and size into the Nature Communications portal (only\npdf allowed). The document was uploaded for us by the submission support and we\nwere not able to validate the document afterwards. Our best guess is that something\nhappened and the photos of the western blot membranes for Fig. 1m, Fig. 2b, Fig. 3b,\nFig. 3i, Fig. 4r, Fig. 4s, Fig.4y, Fig. 5e, Fig. 5f, and Supplementary Fig. 3a that we had\nattached into the corresponding sheets were lost during this process. The source data\nfile with the photos of the western blot membranes is included into this resubmission\nagain.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nWe thank Reviewer for their time co-reviewing our manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "This manuscript reports effects of C1q on human neural stem cells (hNSCs) mediated by the receptor BAI1. The authors describe effects of C1q on hNSC proliferation and other aspects of the cells’ physiology. In parallel studies, the authors provide insight into the signaling pathways that are engaged when C1q binds BAI1, specifically i) regulation of mdm2 to control p53 and ii) endocytosis of C1q to modulate p32. These findings are novel and interesting.\nThe authors were very responsive to the reviewers' comments and have addressed all of the reviewers' concerns following the initial review. The textual changes and new data added by the authors have significantly strengthened the manuscript.", "reviewer_2": "The authors have addressed to the largest extend the queries and concerns raised by this reviewer satisfactorily.\nThe only 'negative' remaining would be that the authors have missed an opportunity pinpoint then intracellular location of the C1q/p32 location better - this was also raised by reviewer 3. I am not sure why as this would have elevated to the work substantially and performing higher resolution/quality staining of mitochondria as suggested by this reviewer (or endosomal compartments as indicated by the authors) is not difficult.\nHowever, this reviewer does do not feel sufficiently strongly about this matter to ask for another revision.", "reviewer_3": "The authors have suitably addressed a number of my concerns and the manuscript has improved substantially. However, a couple of points remain:\n1) I congratulate the authors for carrying out the pull down experiment using each of the interacting proteins. These data are now in Figure 5F. The manner, however, in which these data are presented makes it nearly impossible to interpret. For example, it appears that BAI1 is used as the bait in all lanes (except 3 and 4) shown in these blots and the probed with antibodies above? The presentation of these data could be significantly improved by indicating bait and prey in each case. It took this reviewer, a significant amount of time reviewing the methods and looking at the figure and legend to get an idea of what is going in with that data. The data should be interpretable by simply looking at the figure and legend. In addition, in lanes 3 and 4, it appears that no BAI1 bait was used and yet C1q and p32 are pulled down. How can that be if only BAI1 has the his-tag to bind to the magnetic beads? Is this result of non-specific binding of C1q/p32 to the beads or something else? If the former, this would seriously impact these data and call them into question.\n2) Related to the above, the authors indicate in a number of instances in the response that \"The full original blots are in the Source data.\" This reviewer looked through every file in the manuscript tracking system and could not locate any original blots. These blots would help to interpret the data mentioned in point #1 above.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We appreciate the valuable feedback on our manuscript. We have carefully considered each comment and made revisions accordingly. Additionally, as requested, all of the figures have been formatted to match Nature Communications requirements, and the source data files with raw data have been included in this resubmission. Below, we address each of the points raised."}, {"round": 3, "reviewer_comments": {"reviewer_3": "The authors have suitably addressed my remaining concerns."}, "author_response": ""}]

Decision letter