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Activation of the complement cascade, a powerful effector mechanism of the innate immune system, is associated with neuroinflammation but also with elimination of inappropriate synapses during development. Synthesis of C1q, a recognition component of the complement system, occurs in brain upon ischemia/reperfusion and Alzheimer’s Disease (AD), suggesting that C1q may be a response to injury. In vitro, C1q, in the absence of other complement proteins, improves neuronal viability and neurite outgrowth and prevents Aβ-induced neuronal death suggesting that C1q may have a direct neuroprotective role. Here, investigating the molecular basis for this neuroprotection in vitro, addition of C1q to rat primary cortical neurons significantly up-regulated expression of genes associated with cholesterol metabolism, such as CH25H and INSIG2, and transiently decreased cholesterol levels in neurons, known to facilitate neurite outgrowth. In addition, the expression of syntaxin-3 and its functional association with SNAP25 was increased. C1q also increased the nuclear translocation of CREB and C/EBP-δ, two transcription factors involved in NGF expression and down-regulated specific miRNAs, including let-7c that is predicted to target (and thus inhibit) NGF and NT-3 mRNA. Accordingly, C1q increased expression of NGF and NT-3, and siRNA inhibition of C/EBP-δ, NGF or NT-3 expression prevented the C1q-dependent neurite outgrowth. No such neuroprotective effect is seen in the presence of C3a or C5a. Finally, the induced neuronal gene expression required conformationally intact C1q. These results show that C1q can directly promote neuronal survival thereby demonstrating new interactions between immune proteins and neuronal cells that may facilitate neuroprotection.
The complement system is a powerful effector mechanism of the innate immune system that contributes to protection from infection and resolution of injury (Kohl, 2006). However, tissue damage can result from dysregulated activation of the complement system as seen in arthritis, age-related macular degeneration and Alzheimer’s disease (AD) (Sjoberg et al., 2009;Alexander et al., 2008). Treatment of mouse models of AD with a C5a receptor antagonist significantly reduced neuropathology (Fonseca et al., 2009), suggesting a detrimental consequence of complement activation. However, during development C1q, a component of the complement initiator C1 complex, is expressed in synaptic regions of developing postnatal central nervous system (CNS) and its absence in knock-out mouse results in the failure of anatomical refinement of retinogeniculate connections and excessive retinal innervation (Stevens et al., 2007) and enhanced synaptic connectivity in neocortical slices that lead to epileptogenesis (Chu et al., 2010). Thus, depending on the timing and local environment, the complement cascade can facilitate proper neuronal development or accelerate chronic inflammatory response contributing to neurodegeneration.
Induced synthesis of C1q in the CNS has been seen in several injury models such as viral infection (Dietzschold et al., 1995), kainic acid treatment (Goldsmith et al., 1997), stroke (Huang et al., 1999) and in hippocampal organotypic slice cultures stimulated with Aβ (Fan and Tenner, 2004). In blood, C1q is normally present in complex with proenzymes C1r and C1s as the C1 macromolecular initiator of the classical complement pathway (Ziccardi and Tschopp, 1982). However, C1q can be synthesized in the absence of C1r and C1s by myeloid cells in vitro (Bensa et al., 1983), consistent with the ability of C1q to function not only as part of the C1 complex initiating the classical complement pathway, but independently. C1q in the absence of other complement components has been shown to enhancing ingestion of apoptotic cells by phagocytic cells and modulating inflammation (Fraser et al., 2010;Fraser et al., 2009;Ogden et al., 2001), suggesting a basis for the strong association of lupus with C1q deficiency in humans (Walport et al., 1998). We recently reported that C1q, in the absence of other complement components, increases neuronal survival and neurite outgrowth compared to untreated neurons and protects against Aβ-induced neurotoxicity (Pisalyaput and Tenner, 2008). Heat-inactivated C1q, C1q tails or C1q globular ‘heads’ showed complete loss of neuroprotective ability, indicating that protection requires conformationally intact C1q (Pisalyaput and Tenner, 2008). The observed selective up-regulation of C1q in the CNS and the demonstrated C1q-dependent neuroprotection suggest that C1q can induce a potent and novel neuroprotective program.
Here, microarray analysis has identified candidate molecular mechanisms underlying these novel neuroprotective effects of C1q. In vitro, in primary immature cortical neurons, C1q up-regulated expression of genes associated with cholesterol metabolism and neurite outgrowth and down-regulated the expression of genes associated with inflammation, as well as specific microRNAs (miRNAs), including let-7c that is predicted to target nerve growth factor (NGF) and neurotrophin(NT)-3. The up-regulated expression of several of these genes at the mRNA and protein levels was validated, and suppression of expression of specifically up-regulated genes prevented the C1q-mediated neuronal protection. These results uncover a previously unidentified mechanism of a direct protective effect on neurons of this injury-induced protein thereby revealing novel targets for neuroprotection in the absence of activation of the complement cascade.
Serum-free neurobasal (NB), Trypsin-EDTA, N2 supplement and L-glutamine were obtained from Gibco Life Technologies (Grand Island, NY). Poly-L-lysine hydrobromide, anti-β-actin and microtubule associated protein(MAP)-2 antibodies were obtained from Sigma-Aldrich (St Louis, MO). Anti-NT-3 and NGF-β antibodies were obtained from Millipore (Temecula, CA). Anti-syntaxin (stx)3 and synaptosomal-associated protein (SNAP)25 antibodies were obtained from Abcam (Cambridge, MA). Anti-C/EBP(CCAAT/enhancer-binding protein)-δ, pCREB (cAMP response element-binding protein) and CREB1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa488-conjugated pCREB antibody was obtained from Cell Signaling (Beverly, MA). Human C1q was isolated from serum as previously described (Tenner et al., 1981) and modified by Young et al. (Young et al., 1991). C1q was heat-inactivated for 30 min at 56°C and C1q tails were obtained by pepsin digestion as described (Reid, 1976). Purified human C3a and C5a were obtained from Complement Technology Inc. (Tyler, TX).
All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of University of California Irvine. Cortical neurons were isolated from day 18 Sprague-Dawley rat embryos (Charles River Laboratories, Inc., Wilmington, MA) or day 15 C57BL/6 mouse embryos as previously described (Li et al., 2004). Neurons were plated on poly-L-lysine (1 mg/ml) at a density of 500 cells/mm2 in NB medium supplemented with N2 and grown for 3 days before stimulation with 10 nM C1q. Mouse neurons were treated with 5 nM Ara-C (Sigma-Aldrich) 24 h before adding C1q to limit glia proliferation. In some experiments, neurons were transfected with 5 nM silencer select negative control siRNA (Ambion, Austin, TX) or silencer select siRNA specific for NGF, NT-3 or C/EBP-δ using the siPORT™ NeoFX™ Transfection Agent kit (Ambion), according to manufacturer’s instructions.
Total RNA from untreated neurons and neurons treated with C1q for 3 h was extracted using the RNeasy Mini kit (Qiagen) as previously described (Benoit et al., 2008). RNA quality and quantity were assessed with the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and the NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE). Gene expression profiles were studied using the Rat Gene 1.0 ST array (Affymetrix, Santa Clara, CA). Briefly, total RNA is retro-transcribed to cDNA followed by cRNA amplification and biotin labeling. After purification, cRNA are hybridized on imprinted slides, washed and scanned using the Affymetrix GCOS software (performed by the microarray core facility at University of California, Irvine). Data processing and analysis were performed using JMP Genomics 4.0 software (SAS Institue Inc., Cary, NC). Briefly, inter-array median correction was used to normalize signal intensities. Then, significant differences in gene expression in C1q-treated neurons compared to untreated neurons were identified by ANOVA test using the Bonferroni multiple testing method and a false positive rate (alpha error) of 0.05. Only 388 genes, including 4 miRNAs, with a p-value < 0.01 and an absolute fold difference ≥ 2, were considered as significantly modulated (Supplementary table 1). The EnsEMBL sequence predicted targets of the miRNAs modulated by C1q were determined using the MicroCosm Targets tool of the miRBase, which uses the miRanda algorithm to determine score and the statistical model proposed by Rehmsmeier et al. (Rehmsmeier et al., 2004) to calculate p-value. Only predicted targeted genes with a p-value < 0.001 were included in the analysis. Functional classification and clustering of modulated genes by C1q and miRNA predicted targets were performed using DAVID software (http://david.abcc.ncifcrf.gov/) (Dennis, Jr. et al., 2003;Huang et al., 2009). All data were entered in the Gene Expression Omnibus database following the MIAME procedure (Brazma et al., 2001) and can be retrieved using the accession number GSE18860.
The cDNA synthesis was carried out with 100 ng of total RNA, 0.5μg oligo(dT) primer, 40 units RNaseOUT recombinant ribonuclease inhibitor and 200 units M-MLV reverse transcriptase RT (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. PCR was carried out with 200 ng of cDNA using the Taq Master Mix (QIAGEN). Quantitative PCR was performed using the 7300 fast real-time PCR system (Applied Biosystems, Foster City, CA) and the relative quantification method. Briefly, amplification was conducted in a 25 μl volume using 12.5 μl FastStart SYBR Green Master (ROX) mix (Roche Applied Science, Indianapolis, IN), 100 ng of template cDNA and 0.3 μM each of forward and reverse gene-specific primers. The primers (see Supplementary table 2 for primer sequences) were designed using the primer3 tool (http://frodo.wi.mit.edu/primer3/) and obtained from integrated DNA technologies (IDT DNA, Coralville, IA). RT was omitted in negative controls. The FC in target gene cDNA relative to the GAPDH endogenous control was determined as follows: FC = 2−ΔΔCt, where ΔΔCt = (CtTarget − CtGAPDH)test minus; (CtTarget minus; CtGAPDH)control. Ct values were defined as the number of cycles for which the fluorescence signals were detected (Schmittgen and Livak, 2008).
Neurons were stimulated with 10 nM C1q for different period of times, washed in PBS and harvested with 0.05% Trypsin (Invitrogen). After counting to adjust samples to the same cell number, lipids are extracted in 200 μl chloroform:isopropanol:NP-40 (7:11:0.1). After centrifugation, lipids are dried at 50°C for 30 min followed by a SpeedVac to evaporate the chloroform. Cholesterol levels were determined using the Amplex Red cholesterol assay (Invitrogen) in accordance to the manufacturer’s protocol. Fluorescence was measured using an excitation wavelength of 544 nm and emission wavelength of 590 nm.
Neurons were stimulated with 10 nM C1q for different period of times, fixed with 3.7% paraformaldehyde and permeabilized with 0.1% Triton X-100. For filipin staining, neurons are incubated with 125 μg/ml filipin (Sigma-Aldrich) for 24 h and then washed 3 times. Filipin is a fluorescent polyene antibiotic that forms complexes with cholesterol that can be visualized with ultraviolet light (Schroeder et al., 1971). Immunocytochemistry was performed according to standard procedures adapted from Glynn et al. (Glynn and McAllister, 2006). Briefly, after blocking neurons were incubated with rabbit polyclonal anti-stx3 antibody (dilution 1/100), rabbit polyclonal anti-C/EBP-δ antibody (dilution 1/500), mouse monoclonal anti-MAP-2 antibody (dilution 1/1,000) and/or mouse monoclonal anti-SNAP25 antibody (dilution 1/1,000) for 1 h at room temperature. After 3 washes, slides were incubated with Alexa 488-conjugated anti-rabbit or anti-mouse IgG antibodies (Invitrogen) and Alexa 555-conjugated anti-rabbit or anti-mouse IgG antibodies (Invitrogen) (dilution 1/2,000) for 1 h at room temperature. For pCREB staining, slides were incubated overnight at 4°C with Alexa488-conjugated pCREB antibodies (dilution 1/100). The slides were mounted with 5 μl Prolong Gold anti-fade reagent with DAPI (Invitrogen). Cells were then examined using the Nikon Eclipse Ti-E fluorescent microscope and the NIS-Element AR 3.00, sp7 software. For confocal study, cells were analyzed using the Zeiss LSM710-META confocal microscope and the ZEN2009 software. Protein expression was quantified using Image J software. The nuclear:cytoplasmic ratios of pCREB and C/EBP-δ were quantified as described (Noursadeghi et al., 2008). The total neurite length and the number of roots were determined using NeuronJ (Meijering et al., 2004).
For co-immunoprecipitation experiments, neurons (2.5 million plated in 100 mm dish) were harvested in 500 μl extraction buffer and incubated 15 min on ice. Insoluble fraction is then pelleted by centrifugation at 14,000 rpm for 15 min 4°C. The soluble fraction is incubated with 2.5 μl rabbit anti-stx3 polyclonal antibodies overnight at 4°C under agitation. Then, cell lysates are incubated with 20 μl of protein G Sepharose beads (GE Healthcare) for 2 h at 4°C under agitation. Beads and bound-antibodies were then pelleted by centrifugation (2,000 rpm, 4°C) and washed 3 times in PBS. Pellets are re-suspended in 50 μl loading buffer and boiled 5 min. For soluble protein extraction, neurons were washed with 1 ml cold Hank’s Balanced Salt Solution and harvested in 200 μl of extraction buffer (1% Triton X-100, 25 mM Tris-HCl, 5 mM EDTA, 250 mM NaCl, 10% Glycerol and 1× protease inhibitor cocktail (Roche Applied Science)). After 10 min of incubation on ice, neurons were scraped and the lysate was centrifuged for 15 min at 14,000 rpm at 4°C. The protein concentration in the soluble fraction was determined by microBCA assay (Pierce, Rockford, IL) using bovine serum albumin (BSA) as standards. For Western blot analysis, equal amounts of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes (GE Healthcare, Piscataway, NJ). The membranes were then incubated in blocking buffer (5% BSA/Tris buffer saline(TBS)/Tween 0.1%) for 1 h at room temperature and incubated overnight at 4°C with mouse monoclonal anti-SNAP25 antibody (dilution 1/1,000), rabbit polyclonal anti-NT-3 antibody (1/500), rabbit polyclonal anti-NGF-β antibody (dilution 1/800), rabbit polyclonal anti-pCREB antibody (dilution 1/600), rabbit polyclonal anti-CREB1 antibody (dilution 1/10,000) or mouse monoclonal anti-β-actin antibody (1/2,000). After 3 washes, the membranes were incubated with Horseradish Peroxidase (HRP)-conjugated anti-rabbit (1/5,000, Jackson Immunoresearch Laboratory) or HRP-conjugated anti-mouse (1/10,000, Jackson Immunoresearch Laboratory) antibodies for 1 h at room temperature. The proteins were then developed using enhanced chemiluminescence plus (ECL +, GE Healthcare) and analyzed using the Nikon D700 digital SLR camera and the Image J software as described (Khoury et al., 2010).
Results were calculated as means ± s.d. and compared with two-tailed non parametric Mann-Whitney U test or two-way ANOVA followed by Bonferroni post hoc test, alpha error = 0.05 for all tests. Differences were considered significant when p was < 0.05.
To delineate the C1q-modulated pathways mediating the previously reported in vitro neuroprotection in rat immature neurons (Pisalyaput and Tenner, 2008), we assessed differences in gene expression between untreated and C1q-treated rat primary cortical neurons in vitro by microarray analysis. C1q significantly up-regulated the expression of 127 genes while down-regulating expression of 261 genes as compared to untreated neurons (Supplementary table 1). The microarray results were validated by quantitative real time PCR (16 tested genes, Fig. 1C, note the Y axis is a log2-fold change). The coefficient of correlation between the log2 fold-change (FC) ratio obtained from the microarray and that obtained from the qRT-PCR was 0.84 (data not shown), suggesting that the data obtained from the microarray are highly reliable. Using Gene Ontology (GO) annotation and functional gene clustering, the C1q modulated genes could be classified in 6 major functional groups (Fig. 1A). The first cluster related to cholesterol and lipid metabolism included the up-regulated genes encoding PNPLA7 (also known as NTEL-1 (neuropathy target esterase like 1)), which exhibits an hydrolase activity against lysophospholipid substrates, APOF, which is involved in transport and/or esterification of cholesterol, and INSIG2 and CH25H, which play a role in the regulation of cholesterol homeostasis (Fig. 1A and C). The second cluster associated with membrane and cytoskeleton functions included the up-regulated genes encoding the transmembrane proteins or membrane associated receptors SLC23A2, TMEM79, IL5RA and NT5E (also known as CD73) and proteins involved in membrane expansion, growth of neurites and/or neural cell differentiation such as STX3, SEPT9, CYP26A1 and HPSE. C1q down-regulated the expression of genes associated with neurotransmission (MAOB), chemotaxis (CXCL3 and S100A9), inflammation and cell stress (CASQ2), and development and regulation of cell cycle and death (HEY1 and MESDC2) (Fig. 1A and C). In addition to these 6 main groups, C1q up-regulated the expression of the cyclic adenosine monophosphate (cAMP)-inducible transcription factor C/EBP-δ (Fig. 1C). C1q also modulated the expression of different miRNAs, increasing the expression of the miRNA rno-miR-28 while down-regulating the expression of rno-miR-410, rno-miR-497 and rno-let-7c (Fig. 1B and Supplementary table 1). Predicted targets of these miRNAs are involved in general cellular processes such as metabolic process, regulation of gene expression, transport, signaling and protein modification but also in very specific cellular processes (See Supplementary Fig. 1). Of particular interest, miR-28 is predicted to target some genes associated with regulation of neurotransmission (SYN1) and gene regulation (CREB-L1 and EIF3D). The miRNA miR-410 is predicted to target genes including those associated with signaling (GPR119 and NTRK2 also known as TrkB), while the predicted targets for miR-497 include genes associated with exocytosis (STX1A), regulation of cell death (TNFRSF5, also known as CD40, and AATF, apoptosis-antagonizing transcription factor) and with neurogenesis (ATN-1 and MAP2K1). Finally, the miRNA let-7c is predicted to target genes involved in arginine metabolism (ARG2), regulation of cell death (CLN3) and genes with neurotrophic activities such as NGF, Netrin-1 (NTN1) and NT-3. As miR-410, miR-497 and let-7c are down-regulated by C1q, the expression of their predicted target genes could be predicted to be subsequently increased in C1q-treated neurons.
To test the role of miRNAs in C1q-dependent gene expression, the mRNA levels of miRNA predicted targets was assessed by qRT-PCR over a 16 h time course period (Fig. 1D). As predicted from the C1q-dependent increase expression of miR-28, the expression of the genes predicted to be targeted by miR-28 was generally down-regulated in C1q-treated neurons compared to untreated neurons (Fig. 1D). Furthermore, the expression of the genes predicted to be targeted by miR-410, miR-497 or let-7c, miRNAs that were down-regulated by C1q, was increased generally over the 16 h time course as compared to untreated neurons (Fig. 1D). Together, these data are the first reports demonstrating that C1q modulates expression of genes encoding neurotrophins or involved in regulation of cell death and neurite outgrowth in part through regulation of miRNA expression.
C1q modulated the expression of several enzymes associated with cholesterol metabolism and homeostasis (Fig. 1). To assess the effect on the intracellular cholesterol distribution, neurons were treated or not with C1q for 24 h and then stained with filipin, which forms complexes with cholesterol that can be detected with ultraviolet light (Schroeder et al., 1971). Filipin labelling showed that neurons treated with C1q have similar levels of staining as untreated neurons in the cell body but exhibited a decrease in filipin staining in the neurites as compared to untreated neurons (Fig. 2A) suggesting that the cholesterol distribution is affected by C1q. Since filipin fluorescence intensity is not necessary linearly related to cholesterol content (Maxfield and Wustner, 2002), free cholesterol levels in untreated and C1q-treated neurons was determined by fluorometric assay over a 48 h time course. Neurons treated with C1q showed a very rapid and significant (p < 0.01) decrease in cholesterol content after 4 h of treatment with C1q as compared to untreated neurons (Fig. 2B). The cholesterol content then increased slowly over the 48 h time course in C1q-treated neurons but still significantly lower than levels observed in untreated neurons (Fig. 2B). These results suggest that C1q contributes to regulation of cholesterol distribution and metabolism in neurons.
Stx3 was found to be one of the most up-regulated genes (log2 FC = 3.0, i.e. 8-fold increase, see Supplementary table 1) in C1q-treated neurons (Fig. 1A and C). Immunocytochemical analysis validated the up-regulated stx3 protein expression in neurons stimulated for 24 h with C1q (Fig. 3A and B). C1q-treated neurons showed a 2-fold (p < 0.001) increase in stx3 expression (field area = 14.7 ± 1.0%, Fig. 3B) compared to untreated neurons (field area = 7.0 ± 1.2%, Fig. 3B). Stx3 expression remained significantly (p < 0.05) increased per cell in C1q treated neurons (3.6 ± 0.7, Fig. 3B) compared to untreated neurons (1.9 ± 0.2, Fig. 3B), after Stx3 expression was normalized to the DAPI signal to obviate differences due to a difference in the number of cells between untreated and C1q-treated neurons.
Stx3 stimulates growth of neurites by interacting with SNAP25 (Darios and Davletov, 2006). While SNAP25 expression was not increased in C1q-treated neurons (Supplementary table 1), dual immunostaining of stx3 and SNAP25 clearly demonstrated a strong co-localization of SNAP25 with stx3 (Fig. 3C), particularly in the neurites (Fig. 3C, right panels). Moreover, C1q increased the physical association of SNAP25 with stx3, as seen by the enhanced immunoprecipitation of SNAP25 with stx3 in C1q treated neurons as compared to untreated neurons (Fig. 3D). These results suggest that C1q increases the expression of stx3 protein in neurons potentiating its interaction with SNAP25, its partner required to promote the growth of neurites.
C1q increased the expression of the transcription factor C/EBP-δ (Fig. 1C), which is known to act in concert with CREB to induce NGF expression in the CNS (McCauslin et al., 2006). Since it is known that C1q stimulates the phosphorylation of CREB in monocytes (Fraser et al., 2007), Western blot analysis of neuronal extracts were performed revealing a transient phosphorylation of CREB after 30 min and 1 h of stimulation with C1q (Fig. 4A). Furthermore, using immunocytochemistry (Fig. 4B) and quantitative image analysis (Fig. 4C), we found that the nuclear:cytoplasmic ratio of pCREB was significantly (p < 0.05) increased after 30 min of stimulation with C1q (0.8 ± 0.2) compared to untreated neurons (0.5 ± 0.1). The nuclear translocation of C/EBP-δ in untreated and C1q treated neurons was similarly assessed over a 6 h time course (Fig. 4D and E). C1q significantly increased the nuclear:cytoplasmic C/EBP-δ ratio after 30 min (2.3 ± 0.9 vs. 1.1 ± 0.3, p < 0.05), 1 h (2.6 ± 1.1 vs. 1.0 ± 0.4, p < 0.01) and 6 h (2.3 ± 0.4 vs. 1.1 ± 0.3, p < 0.05) of stimulation as compared to untreated neurons (Fig. 4E). Together, these results show that C1q increased phosphorylation of CREB and nuclear translocation of both pCREB and C/EBP-δ. Dual immunostaining of the two proteins similarly demonstrated that the nuclear co-localization of pCREB and C/EBP-δ is increased after 30 min of stimulation with C1q as compared to untreated neurons (Fig. 4F), consistent with the co-translocation of pCREB and C/EBP-δ to the nucleus.
Finally, specific siRNA that inhibited the expression of C/EBP-δ (Fig. 4G) abrogated the C1q-dependent neuroprotection. As demonstrated by MAP-2 immunocytochemistry (Fig. 4H) and quantitative image analysis (Fig. 4I), C1q significantly (p < 0.05) increased the total neurite length and the number of roots in neurons after 24 h of stimulation. Importantly, inhibition of C/EBP-δ expression in C1q-stimulated neurons significantly decreased both the total neurite length (p < 0.01) and the number of roots (p < 0.05) to levels observed in untreated neurons (Fig. 4I). Together, these data suggest that C/EBP-δ plays a critical role in C1q-triggered neuroprotective pathways and may act in concert with pCREB.
C1q down-regulated the expression of let-7c miRNA, which is predicted to target and thus inhibit the expression of neurotrophic factors, while enhancing C/EBP-Δ expression that can bind and activate neurotrophic factor promoters such as NGF. To determine if neurotrophic factors are indeed modulated in C1q-treated neurons, the expression of NGF and NT-3 in untreated and C1q-treated neurons was specifically assayed by qRT-PCR over a 16 h time course (Fig. 1D). C1q increased the gene expression of NGF (log2 FC = 1.8 ± 0.1) after 6 h of stimulation (Fig. 1D and and5B)5B) and the gene expression of NT-3 after 6 h (log2 FC = 1.9 ± 1.1, Fig. 1D and and5B)5B) and 16 h of stimulation (log2 FC = 2.0 ± 0.6, Fig. 1D) as compared to untreated neurons.
Protein expression of NGF and NT-3 was then assessed by Western blot (Fig. 5A). C1q increased the protein levels of the mature form of NGF after 16 h as compared to untreated neurons, but this increase was transient as NGF levels decreased to untreated levels after 24 h (Fig. 5A). The expression of the NT-3 protein precursor is increased after 24 h and 48 h of culture with C1q (Fig. 5A). These results show that C1q sequentially increased expression of NGF and NT-3 in neurons.
We then assessed the effect of inhibition of NGF and NT-3 on neurotrophin expression and C1q-dependent neuroprotection. Treatment of neurons with specific siRNA targeting NGF resulted in a decrease of NGF mRNA expression in C1q-treated neurons as expected, with no change in NT-3 expression as compared to C1q-stimulated neurons transfected with control siRNA (Fig. 5B). The total neurite length was significantly (p < 0.05) decreased in C1q-stimulated neurons transfected with siNGF compared to control neurons (Fig. 5C and D) at 24 h. The C1q-dependent increased in the number of roots showed a downward trend upon treatment with siNGF but did not reach significance (p = 0.1, Fig. 5D). Interestingly, inhibition of C/EBP-δ expression also prevented the increased in NGF expression after stimulation with C1q (Fig. 5B), suggesting that C/EBP-δ plays a major role in the C1q-dependent up-regulation of NGF. Furthermore, inhibition of NT-3 decreased NT-3 expression in C1q-treated neurons as expected but also resulted in decreased expression of NGF mRNA (Fig. 5B). A blast of siNT-3 sequence against the RNA reference sequence database did not find any match for NGF mRNA sequence (data not shown), ruling out the possibility that the siNT-3 also targeted NGF mRNA and suggesting that NGF expression in C1q-treated neurons is probably due to a direct effect of NT-3. In addition, NT-3 inhibition significantly (p < 0.05) decreased the total neurite length and the number of roots as compared to control neurons (Fig. 5E and F). Together, these results suggest that C1q increased the expression of neurotrophins in neurons and that NGF, under the control of C/EBP-δ and NT-3, plays a critical role in C1q-dependent neuroprotection.
Since C3a and C5a have reported neuroprotective effects in different developmental or environmental contexts (Heese et al., 1998;Osaka et al., 1999;Jauneau et al., 2006;Benard et al., 2008), we evaluated the capacity of C3a and C5a to directly promote neurite outgrowth in this neuron-only culture system (Fig. 6). Neither C3a nor C5a promoted neurite outgrowth (whatever the dose used) (Fig. 6), suggesting that they have no direct neuroprotective effect. Moreover, the neuroprotective effect of C1q is reduced in presence of C3a or C5a in a dose dependent manner (Fig. 6). These results suggest that the direct neuroprotective pathways stimulated by C1q in neurons are fundamentally different than the effects that other complement proteins may have directly or by acting through glia cells.
To validate the generality of what is seen in rat neurons and to enable movement to in vivo mouse models, we determined the effect of C1q on neuronal survival and gene expression in mouse neurons (Fig. 7A and B). As assessed by MAP-2 staining, C1q promoted growth of neurites in mouse neurons (Fig. 7A) as seen in rat neurons (Pisalyaput and Tenner, 2008). In addition, qRT-PCR analysis of gene expression demonstrated that, for all but one of the tested genes, C1q similarly modulates gene expression levels in both rat and mouse neurons (Fig. 7B), implicating similar C1q-stimulated pathways in mouse and rat neurons. Finally, similar to the need for structurally native C1q for the neuroprotective effect (Pisalyaput and Tenner, 2008), the induced gene expression program stimulated by C1q in neurons required a conformationally intact molecule since C1q tails or heat-inactivated C1q failed to promote the induction of STX3, CH25H or C/EBP-δ (Fig. 7C).
In this study, the gene expression and the signaling pathways triggered by C1q in neurons associated with neuroprotection in vitro have been identified by microarray analysis. C1q up-regulated the expression of genes associated with cholesterol and lipid metabolism and membrane and cytoskeleton processes (Fig. 1 and and8),8), two major functions that intrinsically affect organization of neural cell membranes (Piomelli et al., 2007).
C1q modulated the expression of several enzymes associated with cholesterol homeostasis (Fig. 1). Brain derived neurotrophic factor (BDNF), which regulates synaptic function and development, also regulates cholesterol metabolism (Suzuki et al., 2007). Cholesterol depletion in neurons enhances neurite outgrowth (Ko et al., 2005) and treatment of neurons with statins, which are cholesterol-lowering drugs through inhibition of hydroxymethylglutaryl (HMG) CoA (HMG-CoA) reductase, enhances the number of neurites and the neurite length and branching (Pooler et al., 2006). C1q up-regulated the expression of cholesterol-25-hydroxylase (CH25H), an enzyme that catalyzes the conversion of cholesterol to 25-hydroxycholesterol, and the expression of insulin induced gene 2 (INSIG2). Interestingly, INSIG2 and 25-hydroxycholesterol prevent the translocation of sterol regulatory element binding proteins (SREBPs) to the nucleus and thus decrease the transcription of the HMG-CoA reductase, the central enzyme in cholesterol synthesis (Ikonen, 2008;Radhakrishnan et al., 2007). Consistent with the hypothesis that C1q may regulate cholesterol synthesis, both the distribution and levels of cholesterol are affected by C1q in neurons (Fig. 2). The very rapid decrease in cholesterol content after C1q treatment suggests a rapid efflux of cholesterol. Further investigations are needed to determine how C1q modulates cholesterol efflux in neurons but interestingly C1q bound to oxidized (atherogenic) LDL also increases cholesterol efflux in human macrophages (Fraser and Tenner, 2010). C1q upregulated stx3 expression at the mRNA and protein levels and increased its co-localization and interaction with SNAP25 (Fig. 3), a syntaxin partner necessary to promote neurite outgrowth. PC12 neuronal cells completely lacking stx3 are unable to grow neurites, even after stimulation with NGF (Darios and Davletov, 2006). Taken together these data suggest that C1q may enhance neurite outgrowth indirectly through regulation of cholesterol levels and directly through up-regulation of different proteins that promote neurite outgrowth.
C1q treatment resulted in increased phosphorylation of CREB, similar to that seen in monocytes (Fraser et al., 2007), and increased expression of C/EBP-δ (Fig. 4). CREB is a transcription factor critical for long-term memory and synaptic plasticity (Josselyn and Nguyen, 2005). C/EBP proteins have been shown to play a role in synaptic plasticity in the brain (Alberini, 2009). C/EBP-δ and CREB have been shown to both contribute to the inducible expression of NGF in the CNS (Colangelo et al., 1998;McCauslin et al., 2006). Here, addition of C1q increased the nuclear translocation of pCREB and C/EBP-δ with these two factors co-localizing in the nucleus of C1q-treated neurons (Fig. 4). Inhibition of C/EBP-δ expression by siRNA prevented C1q-dependent NGF up-regulation and C1q-dependent neuroprotection (Fig. 4 and and5),5), consistent with a direct role for a C/EBP-δ-NGF cascade in the C1q neuroprotective pathway, with C/EBP-δ as a critical transcription factor in this pathway.
Several miRNAs were also modulated by C1q in neurons (Fig. 1B). MiRNAs function as “guide” molecules in post-transcriptional gene silencing by base pairing with target mRNAs leading to mRNA cleavage or translational repression (Kim, 2005). C1q down regulated miR-410 (a CNS specific miRNA mainly expressed during embryogenesis (Wheeler et al., 2006)), and miR-497 and let-7c, both of which are predicted to target genes involved in regulation of cell death and/or neuronal development and survival. Different miRNAs have been involved in synaptic development, plasticity mechanisms and memory (Kosik, 2006). Members of the murine let-7 family may play a conserved role in synapse development in mammals (Corbin et al., 2009). These results suggest that gene regulation by miRNAs in C1q-treated neurons may be involved in neuronal morphogenesis and/or survival.
As expected from both the down-regulation of let-7c and the nuclear translocation of pCREB and C/EBP-δ, NGF and NT-3 mRNA and protein expression is increased in C1q-treated neurons. The mature form of NGF regulates cell survival while proNGF selectively induces apoptosis (Nykjaer et al., 2004). By increasing protein levels of the mature form of NGF, C1q selectively promotes neuronal survival. Knock-down experiments of NGF and NT-3 using siRNA showed that inhibition of NGF or NT-3 prevents the C1q-dependent neuroprotection (Fig. 5), indicating a critical role for these trophic factors in the C1q-stimulated pathway. We also observed that in presence of siNGF, untreated neurons showed an increase expression of NT-3. NGF may specifically block NT-3 signaling during sympathetic neuron development (Kuruvilla et al., 2004) and reduce NT-3 protein levels in vivo (Randolph et al., 2007), suggesting that the absence of NGF may result in NT-3 increased expression in neurons. Interestingly, inhibition of NT-3 prevented the C1q-induced up-regulation of NGF (Fig. 5B). It has been shown that treatment of Schwann cells with NT-3 increases the expression of NGF and the NGF expression is significantly reduced when cells are cultured in medium containing high glucose which is associated with decreased CREB expression (Suzuki et al., 2004). Thus, NT-3 in concert with CREB, which is phosphorylated and thus activated by C1q in neurons (Fig. 4), contributes to NGF expression.
In AD the presence of the complete C1 complex (C1q plus C1r2C1s2) and the other complement proteins (which are synthesized in the activated/disease state in the CNS) in addition to fibrillar Aβ deposits lead to the activation of the classical complement cascade resulting in the generation of the chemotactic factors C3a and C5a, which can recruit (Yao et al., 1990)) and activate glial cells, and likely synergize with TLR engagement at the plaque (Reed-Geaghan et al., 2009) to stimulate a robust inflammatory reaction including glial secretion of proinflammatory cytokines, reactive oxygen species and nitric oxide, all of which is detrimental to neurons (See model in Figure 8 of (Zhou et al., 2008)). In contrast, other studies have provided evidence for a beneficial role for complement activation products (Wyss-Coray et al., 2002;Maier et al., 2008;Rus et al., 2005), and more recent studies have demonstrated an unexpected role of the early complement cascade C1 through C3 in removing unwanted synapses (i.e. elimination of inappropriate multiple innervation of cortical and retinal neurons) during development (Stevens et al., 2007;Chu et al., 2010). In addition to considering the influence of the local environment on the nature and intensity of the response, it is important to distinguish the activities that result from the activation of the entire or even part of the complement cascade (such as C1 through C3 or C5), from the activities that are induced by the presence of C1q only, i.e. in the absence of any “C1” protease activity (C1r2C1s2) and thus no C3 or C5 convertase activity or other downstream complement activation products (such as C5a or MAC). The higher association of C1q deficiency with lupus (>90%) versus any other complement component in humans (Walport et al., 1998) suggests that there is substantial physiologic consequences of a C1q deficiency, independent of its function as part of the multi component initiator of the classical complement cascade.
Interestingly, C3a, which is produced after complement activation via any of the three activation pathways, has also been shown to induce neuroprotection through NGF expression but mediated through a microglia cell line (Heese et al., 1998) rather than directly on neurons as shown here. More recently, C3a and C5a have been shown to act in synergy with the pro-inflammatory cytokine IL-1β to up-regulate NGF protein in astrocytes (Jauneau et al., 2006), suggesting that C3a and C5a may have indirect neuroprotective effects in vivo, in different developmental or environmental contexts in addition to their noted inflammatory properties in the CNS (Fonseca et al., 2009;Sewell et al., 2004). We showed here that C3a and C5a do not have direct neuroprotective effects and additionally, they reduce the neuroprotective effect of C1q (Fig. 6). Therefore, the ability to increase NGF and NT-3 protein levels directly in this neuron-only culture system with purified C1q protein (no other serum/complement proteins) is fundamentally different from indirect activities of the complement activation products C3a and C5a on neurons/glia cells (reviewed in (Woodruff et al., 2010;Klos et al., 2009)) and is consistent with a previously unappreciated direct neuroprotective response of C1q in the absence of other complement proteins.
In summary, our data demonstrate that C1q triggers a complex program of gene expression that enhances neurite outgrowth and limits neuronal stress and inflammation in vitro (Fig. 8). The induction of C1q synthesis in the absence of other downstream complement proteins both in vitro and early after injury in vivo in several CNS injury models coupled with the subsequent program of gene expression induced by C1q seen here in both rat and mouse neurons, as well as the known ability of C1q alone to enhance clearance of apoptotic cells and suppress inflammatory cytokines (Fraser et al., 2010) suggests a measured, programmed and effective response to injury and a balance between protection and robust inflammation to counteract injury. Thus, while much of the literature has focused on a detrimental effect of immune molecules in neurodegenerative diseases and CNS inflammation, the data presented here emphasize the role of neuro-immune interactions that could be beneficial for neuroprotection. Further analysis of the pathways induced by C1q in response to various in vivo injury models should lead to the discovery of new therapeutic targets to facilitate neuroprotection, proper neurodevelopment and/or restore normal function of the nervous system in AD and other neurodegenerative diseases.
This work was supported by NIH grants AI 41090 and AG 00538, and the Cypress College STEM Summer Bridge Program funded by US Department of Education. The authors thank Rahasson Ager for help and advice with neuron culture preparation and Lindsey Weiner and Ji Young Jang for excellent technical help.