Sans Complement: Amyloid Grows, Synapses and Memory Stay
Crippling the complement cascade in a mouse model of AD leads to more plaques, but less neurodegeneration and better function, according to a new study from Cynthia Lemere and colleagues at Brigham and Women’s Hospital, Boston. Mice missing the complement protein C3 showed an altered immune response to Aβ: Microglia appeared less active, while levels of inflammatory cytokines did not climb as high as in the AD mice with C3. Without C3, synapses were preserved and the mice performed better on tests of learning and memory. The work appeared May 31 in Science Translational Medicine.
“This is a really important paper,” said Andrea Tenner, University of California, Irvine, noting that the rigorous quantitative data support the rationale for investigating therapeutics that inhibit complement activation-dependent processes for the treatment of AD.
Not Attracted: Plaques (green) in APP/PS1 mice lacking C3 (right) have far fewer infiltrating and more peripheral microglia (red) than plaques in C3-replete animals (left). [Courtesy of Science Translational Medicine/AAAS.]
The complement cascade plays a central role in innate immunity. In response to pathogens, dead cells, or debris, C3 and its fragments promote phagocytosis and inflammation. C3 also functions in nervous system development, where it works together with microglia to prune excess synapses, a novel function discovered by co-author Beth Stevens, Children’s Hospital Boston. Stevens and Lemere collaborated to uncover a role for C3 in age-dependent synapse loss in aged mice and in young APP transgenic animals that had yet to accumulate Aβ plaques (see Shi et al., 2015; Apr 2016 news).
What about older mice that have abundant plaques, ongoing complement activation, and inflammation revving up microglia? To address this, first author Qiaoqiao Shi crossed APPsw/PSEN1dE9 mice with C3 knockouts. When offspring were 16 months, old enough to develop plaques and neurodegeneration, she tested their learning and memory skills in a water maze. The C3 knockouts performed better than their C3-replete APP/PS1 controls, and just as well as wild-type mice of the same age. The C3 KOs also had a much higher plaque load than the transgenic controls. Aβ immunoreactivity, thioflavin-S positive plaques, and insoluble Aβ were all greater than in APP/PS1 mice, and the knockouts had many more large plaques.
Nevertheless, neurons fared better in the C3 knockouts. By 16 months, APP/PS1 mice had lost 40 percent of neurons in the CA3 region of the hippocampus, while APP/PS1 C3 knockouts only lost 10 percent. The CA3 is particularly vulnerable to complement-mediated synapse loss, and no neurodegeneration was seen in the CA1, or the dentate gyrus in any of the animals.
The researchers next looked at the astrocytes and microglia to see if their immune response to amyloid plaques might be different in the absence of C3. Indeed, while the numbers of microglia and astrocytes were similar in knockouts and controls, microglia morphology was not. In the knockouts the cells were smaller, with thinner processes, and they had lower levels of activation markers. They also migrated differently. Without complement, strikingly fewer microglia and astrocytes infiltrated the core of plaques, with more hanging back in the outskirts (see image above). “It seems that they come up to the edge but don’t go in,” Lemere said. “Instead of being recruited into the plaque, they seem to be less active and less phagocytic, which is probably why there is more amyloid deposition,” she told Alzforum. In the brain, C3 knockouts also produced less of the inflammatory cytokines TNFα, INFγ, and IL-12.
Lemere sees two possible explanations for the results. One is that C3 knockout suppresses a pro-inflammatory immune response against plaques that would otherwise cause downstream damage to the synapses and neurons. Another is that because plaques in the knockout grow so large, they sequester Aβ oligomers that would normally cause the downstream damage. The two are not mutually exclusive, she said.
However the knockout works, it seems that the mice cope with a higher plaque load, at least until they are 16 months old. “We think that’s because it’s not the plaques themselves that are toxic to the neurons, it’s the immune response to the plaques,” Lemere said.
Previously, Lemere and others reported that knocking out or inhibiting C3 in J20 mice expressing human APP increased not only plaque load but also neurodegeneration (Maier et al., 2008; Wyss-Coray, 2002). The protective effect of C3 knockout in the new study appears to contradict those results. Lemere chalks it up to strain differences in microglial activation. In the J20 mice, glia react less, and show no major alterations after C3 knockout. In the APP/PS1 mice, plaque-induced gliosis actually recapitulates human pathology more closely than in the J20 model, Lemere said.
Shane Liddelow, working in Ben Barres’ lab at Stanford University, agreed that the APP/PS1 mice likely model microglia involvement better than the J20 mice. “I thought the paper was quite interesting. We’ve known for a while that C3 knockouts have increased Aβ plaque load, but Lemere is now showing that even with this increased plaque load, you are seeing beneficial memory outcomes. That is the key finding,” said Liddelow.
Liddelow would like to know more about the glial phenotypes in the AD mice, and especially what the astrocytes do in the absence of C3. He and Barres recently published a study on a novel “killer astrocyte” phenotype, which arises when microglia pump out, among other things, the complement protein C1q (Jan 2017 news). Liddelow pointed out that astrocytes are the main producer of C3, and could be responsible for some of the neurotoxicity seen in these mice.
The results add to a growing enthusiasm for targeting the complement system to modulate inflammation and to treat AD and other neurodegenerative diseases. The mice in the current study never had any C3, so it remains to be seen what happens if C3 is removed or blocked only after older mice have accumulated plaques. Those studies are underway, Lemere told Alzforum. Her lab has a conditional C3 knockout and plans to cross that into the APP/PS1 mice.
William Hu, Emory University, Atlanta, who studies complement activation as a biomarker for AD progression, called the study exciting. “We know that complement activation in spinal fluid seems to be associated with the progression of Alzheimer’s, which presents a very nice human correlate of the phenomenon reported here in mice. What’s so enticing is that complement inhibitors are already approved by the FDA so these could be quickly tested in people,” he told Alzforum.—Pat McCaffrey
- Paper Alert: Microglia Mediate Synaptic Loss in Early Alzheimer’s Disease
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Research Models Citations
- Shi Q, Colodner KJ, Matousek SB, Merry K, Hong S, Kenison JE, Frost JL, Le KX, Li S, Dodart JC, Caldarone BJ, Stevens B, Lemere CA. Complement C3-Deficient Mice Fail to Display Age-Related Hippocampal Decline. J Neurosci. 2015 Sep 23;35(38):13029-42. PubMed.
- Maier M, Peng Y, Jiang L, Seabrook TJ, Carroll MC, Lemere CA. Complement C3 deficiency leads to accelerated amyloid beta plaque deposition and neurodegeneration and modulation of the microglia/macrophage phenotype in amyloid precursor protein transgenic mice. J Neurosci. 2008 Jun 18;28(25):6333-41. PubMed.
- Wyss-Coray T, Yan F, Lin AH, Lambris JD, Alexander JJ, Quigg RJ, Masliah E. Prominent neurodegeneration and increased plaque formation in complement-inhibited Alzheimer's mice. Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10837-42. PubMed.
No Available Further Reading
- Shi Q, Chowdhury S, Ma R, Le KX, Hong S, Caldarone BJ, Stevens B, Lemere CA. Complement C3 deficiency protects against neurodegeneration in aged plaque-rich APP/PS1 mice. Sci Transl Med. 2017 May 31;9(392) PubMed.
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University of California, Irvine
The rigorous quantitative data in this paper support the rationale for investigation of a less-studied group of therapeutics—those inhibiting complement-activation-dependent processes—for treatment of AD and likely for other neurodegenerative disorders, and even aging itself. A main conclusion of this study is that C3-deficiency alters the glial response to amyloid plaques, and subsequently spares cognitive impairment. The data add to the growing awareness that amyloid is necessary but not sufficient for cognitive decline, and that “the reaction of glia to the plaques” is more critical, as is also apparent in human studies. While C3 deficiency protected against hippocampal synapse loss and cognitive flexibility, as the authors point out, either “C3 or its downstream activation fragments such as C3a, C5a and C5b-9 may play an important role in synapse loss and neurodegeneration.” Our previous work (Fonseca, et al., 2009) with a C5a receptor antagonist (i.e., pharmacologic inhibition of a downstream event resulting from complement activation) in two models of AD had an impact on cognitive decline. This current study supports the continuation of targeted investigations of interventions that either work alone, additively, or synergistically with other treatments to prevent cognitive loss. The recent paper from Liddelow and Barres and colleagues showing an upregulation of C3 as a marker for A1 astrocytes in response to one inflammatory signal, LPS, suggests the value of further investigations into whether the induced astrocyte C3 itself via its activation fragments and/or downstream effectors is contributing to the neurotoxicity in these models (Liddelow et al., 2017).
Also notable is the localized/precise quantification of pathology presented in this paper, which highlights the importance of localized changes in the analysis of these models of neurodegeneration (ex. Figure 6, neuron number in CA3 and CA1, and synaptic proteins in hippocampal synaptosomes in Figure 5D). The lack of such extensive analysis in previous studies may contribute to perceived discrepancies between other reports. Kinetic studies correlating pathology, gene expression, neuronal integrity, and behavior will be useful in comparing models and arriving at causative events of dysfunction. This will certainly be a consideration in designing precision human therapeutics, as unnecessarily eliminating the beneficial functions of the complement system in responding to infection and clearance of apoptotic cells and cellular debris should be avoided if possible.
Finally, the authors nicely discuss the limitations of this study, and others, demonstrating the need for future investigations with animal models lacking overexpression of amyloid and presenilin proteins, which will more closely mimic the human sporadic AD condition, as well as the use of inducible and/or conditional KO mice. This very nice paper should be of high impact for the field.
Fonseca MI, Ager RR, Chu SH, Yazan O, Sanderson SD, LaFerla FM, Taylor SM, Woodruff TM, Tenner AJ. Treatment with a C5aR antagonist decreases pathology and enhances behavioral performance in murine models of Alzheimer's disease. J Immunol. 2009 Jul 15;183(2):1375-83. Epub 2009 Jun 26 PubMed.
Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer L, Bennett ML, Münch AE, Chung WS, Peterson TC, Wilton DK, Frouin A, Napier BA, Panicker N, Kumar M, Buckwalter MS, Rowitch DH, Dawson VL, Dawson TM, Stevens B, Barres BA. Neurotoxic reactive astrocytes are induced by activated microglia. Nature. 2017 Jan 26;541(7638):481-487. Epub 2017 Jan 18 PubMed.
Baylor College of Medicine
This paper follows a series of studies by Lemere and colleagues related to the role of complement factor 3 (C3), a central molecule in the complement cascade, in brain aging and Alzheimer’s disease (AD). In particular, the group recently showed that C3 deficiency affords a general protection against age-associated memory decline (Shi et al., 2015). The current report demonstrates a beneficial effect of C3 ablation in rescuing the learning and memory impairment and region-specific neuronal and synapse loss in aged APP/PS1 mice and implicates elevated BDNF/CREB signaling as a potential mechanism. Interestingly, the authors revealed that C3 deficiency results in improved cognitive performance but worsened Aβ pathology. Although the reason for such a contrasting effect and its apparent age-, region-, and possibly strain-dependency is unclear, the data indicates that Aβ load and neuronal and synaptic function can be uncoupled. The protective effect of C3 inactivation is in agreement with our data that aberrant C3 activation, through its cleavage product C3a and interaction with the C3aR, impinges on neuron-glia interaction relevant to AD (Lian et al., 2015; Lian et al., 2016). Overall these findings demonstrate an important role of C3-mediated neuron-immune system cross-talk in AD and support the notion that antagonizing this pathway may be therapeutically beneficial.
Shi Q, Colodner KJ, Matousek SB, Merry K, Hong S, Kenison JE, Frost JL, Le KX, Li S, Dodart JC, Caldarone BJ, Stevens B, Lemere CA. Complement C3-Deficient Mice Fail to Display Age-Related Hippocampal Decline. J Neurosci. 2015 Sep 23;35(38):13029-42. PubMed.
Lian H, Yang L, Cole A, Sun L, Chiang AC, Fowler SW, Shim DJ, Rodriguez-Rivera J, Taglialatela G, Jankowsky JL, Lu HC, Zheng H. NFκB-activated astroglial release of complement C3 compromises neuronal morphology and function associated with Alzheimer's disease. Neuron. 2015 Jan 7;85(1):101-15. Epub 2014 Dec 18 PubMed.
Lian H, Litvinchuk A, Chiang AC, Aithmitti N, Jankowsky JL, Zheng H. Astrocyte-Microglia Cross Talk through Complement Activation Modulates Amyloid Pathology in Mouse Models of Alzheimer's Disease. J Neurosci. 2016 Jan 13;36(2):577-89. PubMed.
University of Southern California
The protein-complement cascade is a major innate immune response mechanism. In this elegant report, Cindy Lemere’s group focused on complement component 3 (C3), which is at the epicenter of the complement cascade and a critical building block for the membrane attack complex (MAC). The authors have dissected a complex network of regulatory factors and have shown that C3 is a multifunctional protein that plays key roles in opsonization of synapses and Aβ, immune modulation, and MAC formation. What’s more, this study shows that the protein complement pathway plays a dominant role in synaptic decline in AD; distinct from changes that occur in normal aging. With respect to the amyloid cascade hypothesis, Aβ-dependent synaptic loss seems entirely complement-driven, once C3 is activated. There is translational value here, too—inhibiting C3 function is a novel therapeutic target to prevent synaptic loss in late onset AD. At the mechanistic level, preventing C3 from activating the MAC would be expected to spare AD-related synaptic loss.
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