Background The classical complement pathway is a complex process involved in the elimination of dead cells, debris, and pathogens, and as well as brain wiring during development. Complement protein C3 is an innate immune molecule central in the complement pathway. It participates in removing pathogens and eliminating synapses, and is elevated with aging and neurodegenerative diseases, including Alzheimer’s Disease. Previously, we showed that lifelong, germline C3‐deficiency protected aged male wild‐type and APPswe/PS1dE9 mice against hippocampal neuronal dysfunction and cognitive decline, despite increasing the Aβ plaque load. However, it remains unknown whether suppressing complement C3 signaling during early stages of AD pathogenesis, when relevant therapeutic interventions might be considered, would confer neuroprotection. Method To address this question, we generated the first‐ever C3 floxed (C3fl/fl) mouse line, and then crossed the C3fl/fl mice with an inducible, global Cre line (Rosa26‐Cre‐ERT2+/‐) for 2 generations to generate novel inducible C3 conditional knockout C3fl/fl; Rosa26‐Cre‐ERT2+/‐ mice. Mice aged 2‐3 months received intraperitoneal injections of either corn oil or tamoxifen (75 mg/kg) once a day for 5 consecutive days. We analyzed the C3 serum levels at 7, 14, 30, 60, and 90 days post‐tamoxifen treatment. We also analyzed the mRNA expression of complement C1q and C3 in the brain and liver 60 days following tamoxifen treatment. Result C3fl/fl;Rosa26‐cre‐ERT2+/‐ mice had a significant 70% reduction in serum C3 levels compared to controls, which was consistent at all timepoints analyzed. We also showed that C3 expression in the liver, the main source of complement proteins, as well as in the brain was reduced, demonstrating effective recombinase activity in these organs. Intriguingly, C1q mRNA expression was increased in the brain and liver following tamoxifen treatment; further studies are underway. Conclusion In conclusion, we present a novel mouse model in which tamoxifen treatment resulted in sustained lowering of C3 in the serum, liver, and brain. We will next cross this model with AD‐like mouse models of amyloidosis and tau pathology to evaluate whether global C3 lowering in early stages AD pathogenesis is protective and if so, our data would support targeting complement as a therapy for AD.
BackgroundComplement C3, a central component of the complement cascade, participates in synaptic elimination during brain development and is elevated in the brain with aging and Alzheimer’s disease (AD). Germline C3 knockout (KO) protects against age‐ and AD‐related hippocampal synapse loss and functional decline (Shi Q et al., J Neurosci 2015; Shi Q et al., Sci Transl Med 2017). We crossed C3fl/fl mice with Rosa26‐Cre‐ERT2 mice to generate global C3 inducible conditional KO mice (C3iKO). Previously, we reported that 5 daily TAM treatments in C3iKO mice at 4‐5 mo of age led to sustained C3 reductions of ∼95% in serum and ∼80% in brain, and protected mice against age‐related cognitive decline at 16‐17 months. In addition, TAM treatment at 3‐4 mo of age protected long‐term potentiation (LTP) in hippocampal slices exposed to toxic Aβ dimers (S26C) obtained at 7‐8 mo of age. Here, we extend our analyses to include changes in complement, cytokine and ApoE expression in these same C3iKO mice and provide confirmatory results in a second cohort of mice.MethodqRT‐PCR was used to quantify brain gene expression in the first cohort. In the second cohort, C3iKO, C3fl/fl and C3KO mice were treated with TAM or corn oil (CO) at 7‐8 mo of age. Cognitive testing commenced at 17‐18 mo and included the Spatial Novelty Y Maze (SPNY), Novel Object Recognition (NOR) and Displaced Object Recognition (DOR). Mice were euthanized and hippocampal synaptosomes isolated for Western blot analysis.ResultOne‐year post‐treatment, C1qa, C1qb, C1qc, C3, CD11b, CD18, IL‐1β, IFNα, IFNβ, and APOE mRNAs were reduced, and TGFβ and TGFβR1 mRNAs were elevated in brains of TAM‐treated C3iKO and C3KO mice compared to CO‐treated C3iKO mice. In the second cohort, C3 protein levels were reduced ∼92% in serum and ∼77% in hippocampal synaptosomes in TAM‐treated C3iKO mice. TAM‐treated C3iKO mice and both C3KO groups showed significantly better performance in SNYM, LOR and DOR tests compared to controls, consistent with our first study. Pre‐ and post‐synaptic markers were elevated in TAM‐treated vs. CO‐treated C3iKO hippocampal synaptosomes.ConclusionGlobal C3 lowering in adulthood has long‐lasting effects on immune signaling and hippocampal function. (NIH RF1 AG060057‐CAL)
Background Complement component C3, an innate immune molecule, is important for removing pathogens and eliminating synapses during brain development, aging, and Alzheimer’s disease (AD). Previously, our group demonstrated that C3 deficiency protected against synapse loss during normal aging in wild‐type and APPswe/PS1dE9 mice. Induction of C3 deficiency after normal brain development will help inform whether its inhibition in aging or early stages of AD may serve as a future therapeutic strategy. Method We crossed C3fl/fl mice with an inducible, global Cre line (Rosa26‐Cre‐ERT2+/‐) for 2 generations to generate novel inducible C3 conditional knockout C3fl/fl; Rosa26‐Cre‐ERT2+/‐ mice (C3iKO). In one cohort, four‐to‐five‐month‐old mice were injected with tamoxifen (TAM) or corn oil (CO) daily for 5 days. Behavioral tasks were performed when these mice reached 16‐17 months of age. In another study, three‐to‐four‐month‐old female mice were injected with either CO/TAM and electrophysiological recording of long‐term potentiation (LTP) was conducted in hippocampal slices of TAM‐treated and CO‐treated mice at 7‐8 months of age following incubation of the slices with neurotoxic Aβ S26 dimers. Result C3iKO mice had a significant 85‐95% reduction in serum C3 levels compared to controls, which was consistent at all timepoints analyzed (from Day 7 to Day 365). In this cohort, behavioral testing for hippocampal‐dependent spatial memory, object memory, and object location was performed when TAM‐treated and CO‐treated mice reached 16‐17 months of age. C3iKO+TAM mice performed significantly better than C3iKO+CO‐treated mice in these behavioral tasks, indicating that C3 lowering after brain development protected mice from age‐related cognitive decline. In the second study, we found that C3 lowering in adult mice protected hippocampal synapses from Aβ S26 dimer‐mediated LTP impairment. Conclusion Global C3 depletion in C3iKO young adult mice protected against hippocampal dysfunction as they aged, suggesting that C3 lowering may be an effective therapeutic strategy for aging and possibly, AD. Future studies are underway to investigate the C3‐mediated mechanisms of synaptic dysfunction in the hippocampus.
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