C1q-induced LRP1B and GPR6 proteins expressed early in Alzheimer disease mouse models, are essential for the C1q-mediated protection against amyloid-ß neurotoxicity.

Complement protein C1q is induced in the brain in response to a variety of neuronal injuries, including Alzheimer disease (AD), and blocks fibrillar amyloid-ß (fAß) neurotoxicity in vitro. Here, we show that C1q protects immature and mature primary neurons against fAß toxicity, and we report for the first time that C1q prevents toxicity induced by oligomeric forms of amyloid-ß (Aß). Gene expression analysis reveals C1q-activated phosphorylated cAMP-response element-binding protein and AP-1, two transcription factors associated with neuronal survival and neurite outgrowth, and increased LRP1B and G protein-coupled receptor 6(GPR6) expression in fAß-injured neurons. Silencing of cAMP-response element-binding protein, LRP1B or GPR6 expression inhibited C1q-mediated neuroprotection from fAß-induced injury. In addition, C1q altered the association of oligomeric Aß and fAß with neurons. In vivo, increased hippocampal expression of C1q, LRP1B, and GPR6 is observed as early as 2 months of age in the 3 × Tg mouse model of AD, whereas no such induction of LRP1B and GPR6 was seen in C1q-deficient AD mice. In contrast, expression of C1r and C1s, proteases required to activate the classical complement pathway, and C3 showed a significant age-dependent increase only after 10-13 months of age when Aß plaques start to accumulate in this AD model. Thus, our results identify pathways by which C1q, up-regulated in vivo early in response to injury without the coordinate induction of other complement components, can induce a program of gene expression that promotes neuroprotection and thus may provide protection against Aß in preclinical stages of AD and other neurodegenerative processes.

Complement protein C1q directs macrophage polarization and limits inflammasome activity during the uptake of apoptotic cells.

Deficiency in C1q, the recognition component of the classical complement cascade and a pattern recognition receptor involved in apoptotic cell clearance, leads to lupus-like autoimmune diseases characterized by auto-antibodies to self proteins and aberrant innate immune cell activation likely due to impaired clearance of apoptotic cells. In this study, we developed an autologous system using primary human lymphocytes and human monocyte-derived macrophages (HMDMs) to characterize the effect of C1q on macrophage gene expression profiles during the uptake of apoptotic cells. C1q bound to autologous apoptotic lymphocytes modulated expression of genes associated with JAK/STAT signaling, chemotaxis, immunoregulation, and NLRP3 inflammasome activation in LPS-stimulated HMDMs. Specifically, C1q sequentially induced type I IFNs, IL-27, and IL-10 in LPS-stimulated HMDMs and IL-27 in HMDMs when incubated with apoptotic lymphocyte conditioned media. Coincubation with C1q tails prevented the induction of type I IFNs and IL-27 in a dose-dependent manner, and neutralization of type I IFNs partially prevented IL-27 induction by C1q. Finally, C1q decreased procaspase-1 cleavage and caspase-1-dependent cleavage of IL-1ß suggesting a potent inhibitory effect of C1q on inflammasome activation. These results identify specific molecular pathways induced by C1q to suppress macrophage inflammation and provide potential therapeutic targets to control macrophage polarization and thus inflammation and autoimmunity.

Complement protein C1q-mediated neuroprotection is correlated with regulation of neuronal gene and microRNA expression.

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 during ischemia/reperfusion and Alzheimer's disease, 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 ß-amyloid-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 upregulated expression of genes associated with cholesterol metabolism, such as cholesterol-25-hydroxylase and insulin induced gene 2, and transiently decreased cholesterol levels in neurons, known to facilitate neurite outgrowth. In addition, the expression of syntaxin-3 and its functional association with synaptosomal-associated protein 25 was increased. C1q also increased the nuclear translocation of cAMP response element-binding protein and CCAAT/enhancer-binding protein-δ (C/EBP-δ), two transcription factors involved in nerve growth factor (NGF) expression and downregulated specific microRNAs, including let-7c that is predicted to target (and thus inhibit) NGF and neurotrophin-3 (NT-3) mRNA. Accordingly, C1q increased expression of NGF and NT-3, and small interfering RNA 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.

Contribution of complement activation pathways to neuropathology differs among mouse models of Alzheimer's disease.

BACKGROUND: Complement proteins and activation products have been found associated with neuropathology in Alzheimer's disease (AD). Recently, a C5a receptor antagonist was shown to suppress neuropathology in two murine models of AD, Tg2576 and 3xTg. Previously, a genetic deficiency of C1q in the Tg2576 mouse model showed an accumulation of fibrillar plaques similar to the complement sufficient Tg2576, but reactive glia were significantly decreased and neuronal integrity was improved suggesting detrimental consequences for complement activation in AD. The goal of this study was to define the role of the classical complement activation pathway in the progression of pathology in the 3xTg mouse that develops tangles in addition to fibrillar plaques (more closely reflecting human AD pathology) and to assess the influence of complement in a model of AD with a higher level of complement hemolytic activity. METHODS: 3xTg mice deficient in C1q (3xTgQ-/-) were generated, and both 3xTg and 3xTgQ-/- were backcrossed to the BUB mouse strain which has higher in vitro hemolytic complement activity. Mice were aged and perfused, and brain sections stained for pathological markers or analyzed for proinflammatory marker expression. RESULTS: 3xTgQ-/- mice showed similar amounts of fibrillar amyloid, reactive glia and hyperphosphorylated tau as the C1q-sufficient 3xTg at the ages analyzed. However, 3xTg and 3xTgQ-/- on the BUB background developed pathology earlier than on the original 3xTg background, although the presence of C1q had no effect on neuropathological and pro-inflammatory markers. In contrast to that seen in other transgenic models of AD, C1q, C4 and C3 immunoreactivity was undetectable on the plaques of 3xTg in any background, although C3 was associated with reactive astrocytes surrounding the plaques. Importantly, properdin a component of the alternative complement pathway was associated with plaques in all models. CONCLUSIONS: In contrast to previously investigated transgenic models of AD, development of neuropathology in 3xTg mice, which progresses much slower than other murine models, may not be influenced by fibrillar amyloid mediated activation of the classical complement pathway, suggesting that the alternative complement pathway activation or a C3-independent cleavage of C5 could account for the detrimental effects in these mice that are prevented by the C5a receptor antagonist. Furthermore, the paucity of complement activation may be a factor in the slower kinetics of progression of pathology in the 3xTg model of this disease.

C1q Binding to and Uptake of Apoptotic Lymphocytes by Human Monocyte-derived Macrophages.

To characterize macrophage gene expression profiles during the uptake of autologous apoptotic cells, we developed a unique, more physiologic system using primary human monocyte derived macrophages purified via a nonactivating isolation procedure (and in the absence of contaminating platelets, which can release stimulating signals if activated) and autologous lymphocytes as a source of apoptotic cells. The use of autologous cells as the apoptotic target rather than transformed cell lines avoids antigenic stimulation from "nonself" structures at the HLA level but also from "altered self" signals due to the transformation inherent in cell lines.

Purification of Human Monocytes and Lymphocyte Populations by Counter Current Elutriation- A Short Protocol.

Investigations of the activation processes involved in human monocytes and monocyte-derived macrophages and dendritic cells often required large numbers of cells that have not been possibly altered or activated by adherence to surfaces, by binding of antibodies to surface antigens during positive selection, or by release of activators by platelets or other non myeloid cells during isolation or co-culture. Human peripheral blood monocytes as well as lymphocytes from the same blood donor can be isolated by counterflow elutriation using a modification of the technique of Lionetti et al., 1980 as described previously (Bobak et al., 1986). From a unit of blood drawn into anticoagulant, 60-120 million monocytes can be obtained. These cells are not activated and have been shown to be appropriately capable of differential activation in multiple studies.