C5aR1 signaling promotes region- and age-dependent synaptic pruning in models of Alzheimer's disease.

INTRODUCTION: Synaptic loss is a hallmark of Alzheimer's disease (AD) that correlates with cognitive decline in AD patients. Complement-mediated synaptic pruning has been associated with this excessive loss of synapses in AD. Here, we investigated the effect of C5aR1 inhibition on microglial and astroglial synaptic pruning in two mouse models of AD. METHODS: A combination of super-resolution and confocal and tridimensional image reconstruction was used to assess the effect of genetic ablation or pharmacological inhibition of C5aR1 on the Arctic48 and Tg2576 models of AD. RESULTS: Genetic ablation or pharmacological inhibition of C5aR1 partially rescues excessive pre-synaptic pruning and synaptic loss in an age and region-dependent fashion in two mouse models of AD, which correlates with improved long-term potentiation (LTP). DISCUSSION: Reduction of excessive synaptic pruning is an additional beneficial outcome of the suppression of C5a-C5aR1 signaling, further supporting its potential as an effective targeted therapy to treat AD. HIGHLIGHTS: C5aR1 ablation restores long-term potentiation in the Arctic model of AD. C5aR1 ablation rescues region specific excessive pre-synaptic loss. C5aR1 antagonist, PMX205, rescues VGlut1 loss in the Tg2576 model of AD. C1q tagging is not sufficient to induce VGlut1 microglial ingestion. Astrocytes contribute to excessive pre-synaptic loss at late stages of the disease.

C5aR1 signaling promotes region and age dependent synaptic pruning in models of Alzheimer's Disease.

INTRODUCTION: Synaptic loss is a hallmark of Alzheimer's disease (AD) that correlates with cognitive decline in AD patients. Complement-mediated synaptic pruning has been associated with this excessive loss of synapses in AD. Here, we investigated the effect of C5aR1 inhibition on microglial and astroglial synaptic pruning in two mouse models of AD. METHODS: A combination of super-resolution and confocal and tridimensional image reconstruction was used to assess the effect of genetic ablation or pharmacological inhibition of C5aR1 on the Arctic48 and Tg2576 models of AD. RESULTS: Genetic ablation or pharmacological inhibition of C5aR1 rescues the excessive pre-synaptic pruning and synaptic loss in an age and region dependent fashion in two mouse models of AD, which correlates with improved long-term potentiation (LTP). DISCUSSION: Reduction of excessive synaptic pruning is an additional beneficial outcome of the suppression of C5a-C5aR1 signaling, further supporting its potential as an effective targeted therapy to treat AD.

Influence of complement protein C1q or complement receptor C5aR1 on gut microbiota composition in wildtype and Alzheimer's mouse models.

The contribution of the gut microbiome to neuroinflammation, cognition, and Alzheimer's disease progression has been highlighted over the past few years. Additionally, inhibition of various components of the complement system has repeatedly been demonstrated to reduce neuroinflammation and improve cognitive performance in AD mouse models. Whether the deletion of these complement components is associated with distinct microbiome composition, which could impact neuroinflammation and cognitive performance in mouse models has not yet been examined. Here, we provide a comprehensive analysis of conditional and constitutive knockouts, pharmacological inhibitors, and various housing paradigms for the animal models and wild-type controls at various ages. We aimed to determine the impact of C1q or C5aR1 inhibition on the microbiome in the Arctic and Tg2576 mouse models of AD, which develop amyloid plaques at different ages and locations. Analysis of fecal samples from WT and Arctic mice following global deletion of C1q demonstrated significant alterations to the microbiomes of Arctic but not WT mice, with substantial differences in abundances of Erysipelotrichales, Clostridiales and Alistipes. While no differences in microbiome diversity were detected between cohoused wildtype and Arctic mice with or without the constitutive deletion of the downstream complement receptor, C5aR1, a difference was detected between the C5aR1 sufficient (WT and Arctic) and deficient (C5ar1KO and ArcticC5aR1KO) mice, when the mice were housed segregated by C5aR1 genotype. However, cohousing of C5aR1 sufficient and deficient wildtype and Arctic mice resulted in a convergence of the microbiomes and equalized abundances of each identified order and genus across all genotypes. Similarly, pharmacologic treatment with the C5aR1 antagonist, PMX205, beginning at the onset of beta-amyloid plaque deposition in the Arctic and Tg2576 mice, demonstrated no impact of C5aR1 inhibition on the microbiome. This study demonstrates the importance of C1q in microbiota homeostasis in neurodegenerative disease. In addition, while demonstrating that constitutive deletion of C5aR1 can significantly alter the composition of the fecal microbiome, these differences are not present when C5aR1-deficient mice are cohoused with C5aR1-sufficient animals with or without the AD phenotype and suggests limited if any contribution of the microbiome to the previously observed prevention of cognitive and neuronal loss in the C5aR1-deficient AD models.

C5aR1 antagonism suppresses inflammatory glial gene expression and alters cellular signaling in an aggressive Alzheimer's model.

Alzheimer's disease (AD) is the leading cause of dementia in older adults, and the need for effective, sustainable therapeutic targets is imperative. Pharmacologic inhibition of C5aR1 reduces plaque load, gliosis and memory deficits in animal models. However, the cellular basis underlying this neuroprotection and which processes were the consequence of amyloid reduction vs alteration of the response to amyloid were unclear. In the Arctic model, the C5aR1 antagonist PMX205 did not reduce plaque load, but deficits in short-term memory in female mice were prevented. Hippocampal single cell and single nucleus RNA-seq clusters revealed C5aR1 dependent and independent gene expression and cell-cell communication. Microglial clusters containing neurotoxic disease-associated microglial genes were robustly upregulated in Arctic mice and drastically reduced with PMX205 treatment, while genes in microglia clusters that were overrepresented in the Arctic-PMX205 vs Arctic group were associated with synapse organization and transmission and learning. PMX205 treatment also reduced some A-1 astrocyte genes. In spite of changes in transcript levels, overall protein levels of some reactive glial markers were relatively unchanged by C5aR1 antagonism, as were clusters associated with protective responses to injury. C5aR1 inhibition promoted signaling pathways associated with cell growth and repair, such as TGFß and FGF, in Arctic mice, while suppressing inflammatory pathways including PROS, Pecam1, and EPHA. In conclusion, pharmacologic C5aR1 inhibition prevents cognitive loss, limits microglial polarization to a detrimental inflammatory state and permits neuroprotective responses, as well as leaving protective functions of complement intact, making C5aR1 antagonism an attractive therapeutic strategy for individuals with AD.

C5aR1 antagonism alters microglial polarization and mitigates disease progression in a mouse model of Alzheimer's disease.

Multiple studies have recognized the involvement of the complement cascade during Alzheimer's disease pathogenesis. However, the specific role of C5a-C5aR1 signaling in the progression of this neurodegenerative disease is still not clear. Furthermore, its potential as a therapeutic target to treat AD still remains to be elucidated. Canonically, generation of the anaphylatoxin C5a as the result of complement activation and interaction with its receptor C5aR1 triggers a potent inflammatory response. Previously, genetic ablation of C5aR1 in a mouse model of Alzheimer's disease exerted a protective effect by preventing cognitive deficits. Here, using PMX205, a potent, specific C5aR1 antagonist, in the Tg2576 mouse model of Alzheimer's disease we show a striking reduction in dystrophic neurites in parallel with the reduced amyloid load, rescue of the excessive pre-synaptic loss associated with AD cognitive impairment and the polarization of microglial gene expression towards a DAM-like phenotype that are consistent with the neuroprotective effects seen. These data support the beneficial effect of a pharmacological inhibition of C5aR1 as a promising therapeutic approach to treat Alzheimer's disease. Supportive of the safety of this treatment is the recent FDA-approval of another other C5a receptor 1 antagonist, Avacopan, as a treatment for autoimmune inflammatory diseases.

Modulation of C5a-C5aR1 signaling alters the dynamics of AD progression.

BACKGROUND: The complement system is part of the innate immune system that clears pathogens and cellular debris. In the healthy brain, complement influences neurodevelopment and neurogenesis, synaptic pruning, clearance of neuronal blebs, recruitment of phagocytes, and protects from pathogens. However, excessive downstream complement activation that leads to generation of C5a, and C5a engagement with its receptor C5aR1, instigates a feed-forward loop of inflammation, injury, and neuronal death, making C5aR1 a potential therapeutic target for neuroinflammatory disorders. C5aR1 ablation in the Arctic (Arc) model of Alzheimer's disease protects against cognitive decline and neuronal injury without altering amyloid plaque accumulation. METHODS: To elucidate the effects of C5a-C5aR1 signaling on AD pathology, we crossed Arc mice with a C5a-overexpressing mouse (ArcC5a+) and tested hippocampal memory. RNA-seq was performed on hippocampus and cortex from Arc, ArcC5aR1KO, and ArcC5a+ mice at 2.7-10 months and age-matched controls to assess mechanisms involved in each system. Immunohistochemistry was used to probe for protein markers of microglia and astrocytes activation states. RESULTS: ArcC5a+ mice had accelerated cognitive decline compared to Arc. Deletion of C5ar1 delayed or prevented the expression of some, but not all, AD-associated genes in the hippocampus and a subset of pan-reactive and A1 reactive astrocyte genes, indicating a separation between genes induced by amyloid plaques alone and those influenced by C5a-C5aR1 signaling. Biological processes associated with AD and AD mouse models, including inflammatory signaling, microglial cell activation, and astrocyte migration, were delayed in the ArcC5aR1KO hippocampus. Interestingly, C5a overexpression also delayed the increase of some AD-, complement-, and astrocyte-associated genes, suggesting the possible involvement of neuroprotective C5aR2. However, these pathways were enhanced in older ArcC5a+ mice compared to Arc. Immunohistochemistry confirmed that C5a-C5aR1 modulation in Arc mice delayed the increase in CD11c-positive microglia, while not affecting other pan-reactive microglial or astrocyte markers. CONCLUSION: C5a-C5aR1 signaling in AD largely exerts its effects by enhancing microglial activation pathways that accelerate disease progression. While C5a may have neuroprotective effects via C5aR2, engagement of C5a with C5aR1 is detrimental in AD models. These data support specific pharmacological inhibition of C5aR1 as a potential therapeutic strategy to treat AD.

Incorporation of a Ligand Peptide for Immune Inhibitory Receptor LAIR-1 on Biomaterial Surfaces Inhibits Macrophage Inflammatory Responses.

Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is an inhibitory receptor broadly expressed on immune cells, with its ligands residing within the extracellular matrix protein collagen. In this study, surfaces are modified with a LAIR-1 ligand peptide (LP), and it is observed that macrophages cultured on LAIR-1 LP-conjugated surfaces exhibit significantly reduced secretion of inflammatory cytokines in response to proinflammatory stimuli that reflect an injured environment. These downregulated mediators include TNF-α, MIP-1α, MIP-1ß, MIP-2, RANTES, and MIG. Knockdown of LAIR-1 using siRNA abrogates this inhibition of cytokine secretion, supporting the specificity of the inhibitory effect to this receptor. These results are the first to demonstrate that integration of LAIR-1 ligands with biomaterials could suppress inflammatory responses.

Prevention of C5aR1 signaling delays microglial inflammatory polarization, favors clearance pathways and suppresses cognitive loss.

BACKGROUND: Pharmacologic inhibition of C5aR1, a receptor for the complement activation proinflammatory fragment, C5a, suppressed pathology and cognitive deficits in Alzheimer's disease (AD) mouse models. To validate that the effect of the antagonist was specifically via C5aR1 inhibition, mice lacking C5aR1 were generated and compared in behavior and pathology. In addition, since C5aR1 is primarily expressed on cells of the myeloid lineage, and only to a lesser extent on endothelial cells and neurons in brain, gene expression in microglia isolated from adult brain at multiple ages was compared across all genotypes. METHODS: C5aR1 knock out mice were crossed to the Arctic AD mouse model, and characterized for pathology and for behavior performance in a hippocampal dependent memory task. CX3CR1GFP and CCR2RFP reporter mice were bred to C5aR1 sufficient and knockout wild type and Arctic mice to enable sorting of microglia (GFP-positive, RFP-negative) isolated from adult brain at 2, 5, 7 and 10 months of age followed by RNA-seq analysis. RESULTS: A lack of C5aR1 prevented behavior deficits at 10 months, although amyloid plaque load was not altered. Immunohistochemical analysis showed no CCR2+ monocytes/macrophages near the plaques in the Arctic brain with or without C5aR1. Microglia were sorted from infiltrating monocytes (GFP and RFP-positive) for transcriptome analysis. RNA-seq analysis identified inflammation related genes as differentially expressed, with increased expression in the Arctic mice relative to wild type and decreased expression in the Arctic/C5aR1KO relative to Arctic. In addition, phagosomal-lysosomal gene expression was increased in the Arctic mice relative to wild type but further increased in the Arctic/C5aR1KO mice. A decrease in neuronal complexity was seen in hippocampus of 10 month old Arctic mice at the time that correlates with the behavior deficit, both of which were rescued in the Arctic/C5aR1KO. CONCLUSIONS: These data are consistent with microglial polarization in the absence of C5aR1 signaling reflecting decreased induction of inflammatory genes and enhancement of degradation/clearance pathways, which is accompanied by preservation of CA1 neuronal complexity and hippocampal dependent cognitive function. These results provide links between microglial responses and loss of cognitive performance and, combined with the previous pharmacological approach to inhibit C5aR1 signaling, support the potential of this receptor as a novel therapeutic target for AD in humans.

Cell-specific deletion of C1qa identifies microglia as the dominant source of C1q in mouse brain.

BACKGROUND: The complement cascade not only provides protection from infection but can also mediate destructive inflammation. Complement is also involved in elimination of neuronal synapses which is essential for proper development, but can be detrimental during aging and disease. C1q, required for several of these complement-mediated activities, is present in the neuropil, microglia, and a subset of interneurons in the brain. METHODS: To identify the source(s) of C1q in the brain, the C1qa gene was selectively inactivated in the microglia or Thy-1+ neurons in both wild type mice and a mouse model of Alzheimer's disease (AD), and C1q synthesis assessed by immunohistochemistry, QPCR, and western blot analysis. RESULTS: While C1q expression in the brain was unaffected after inactivation of C1qa in Thy-1+ neurons, the brains of C1qa FL/FL :Cx3cr1 CreERT2 mice in which C1qa was ablated in microglia were devoid of C1q with the exception of limited C1q in subsets of interneurons. Surprisingly, this loss of C1q occurred even in the absence of tamoxifen by 1 month of age, demonstrating that Cre activity is tamoxifen-independent in microglia in Cx3cr1 CreERT2/WganJ mice. C1q expression in C1qa FL/FL : Cx3cr1 CreERT2/WganJ mice continued to decline and remained almost completely absent through aging and in AD model mice. No difference in C1q was detected in the liver or kidney from C1qa FL/FL : Cx3cr1 CreERT2/WganJ mice relative to controls, and C1qa FL/FL : Cx3cr1 CreERT2/WganJ mice had minimal, if any, reduction in plasma C1q. CONCLUSIONS: Thus, microglia, but not neurons or peripheral sources, are the dominant source of C1q in the brain. While demonstrating that the Cx3cr1 CreERT2/WganJ deleter cannot be used for adult-induced deletion of genes in microglia, the model described here enables further investigation of physiological roles of C1q in the brain and identification of therapeutic targets for the selective control of complement-mediated activities contributing to neurodegenerative disorders.

The AVRDC - The World Vegetable Center mungbean (Vigna radiata) core and mini core collections.

BACKGROUND: Large ex situ germplasm collections generally harbor a wide range of crop diversity. AVRDC--The World Vegetable Center is holding in trust the world's second largest mungbean (Vigna radiata) germplasm collection with more than 6,700 accessions. Screening large collections for traits of interest is laborious and expensive. To enhance the access of breeders to the diversity of the crop, mungbean core and mini core collections have been established. RESULTS: The core collection of 1,481 entries has been built by random selection of 20% of the accessions after geographical stratification and subsequent cluster analysis of eight phenotypic descriptors in the whole collection. Summary statistics, especially the low differences of means, equal variance of the traits in both the whole and core collection and the visual inspection of quantile-quantile plots comparing the variation of phenotypic traits present in both collections indicated that the core collection well represented the pattern of diversity of the whole collection. The core collection was genotyped with 20 simple sequence repeat markers and a mini core set of 289 accessions was selected, which depicted the allele and genotype diversity of the core collection. CONCLUSIONS: The mungbean core and mini core collections plus their phenotypic and genotypic data are available for distribution to breeders. It is expected that these collections will enhance the access to biodiverse mungbean germplasm for breeding.

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.

Microglial C5aR (CD88) expression correlates with amyloid-beta deposition in murine models of Alzheimer's disease.

Alzheimer's disease (AD), a progressive neurodegenerative disease characterized by the accumulation of amyloid-beta protein and neuronal loss, is the leading cause of age-related dementia in the world today. The disease is also associated with neuroinflammation, robust activation of astrocytes and microglia, and evidence of activation of the complement system, localized with both fibrillar amyloid-beta (fAbeta) plaques and tangles. The observations are consistent with a complement-dependent component of AD progression. We have previously shown that inhibition of the major complement receptor for C5a (CD88) with the antagonist PMX205 results in a significant reduction in pathology in two mouse models of AD. To further characterize the role of complement in AD-related neuroinflammation, we examined the age- and disease-associated expression of CD88 in brain of transgenic mouse models of AD and the influence of PMX205 on the presence of various complement activation products using flow cytometry, western blot, and immunohistochemistry. CD88 was found to be up-regulated in microglia, in the immediate vicinity of amyloid plaques. While thioflavine plaque load and glial recruitment is significantly reduced after treatment with PMX205, C1q remains co-localized with fAbeta plaques and C3 is still expressed by the recruited astrocytes. Thus, with PMX205, potentially beneficial activities of these early complement components may remain intact, while detrimental activities resulting from C5a-CD88 interaction are inhibited. This further supports the targeted inhibition of specific complement mediated activities as an approach for AD therapy.

Treatment with a C5aR antagonist decreases pathology and enhances behavioral performance in murine models of Alzheimer's disease.

Alzheimer's disease (AD) is an age-related dementia, characterized by amyloid plaques, neurofibrillary tangles, neuroinflammation, and neuronal loss in the brain. Components of the complement system, known to produce a local inflammatory reaction, are associated with the plaques and tangles in AD brain, and thus a role for complement-mediated inflammation in the acceleration or progression of disease has been proposed. A complement activation product, C5a, is known to recruit and activate microglia and astrocytes in vitro by activation of a G protein-coupled cell-surface C5aR. Here, oral delivery of a cyclic hexapeptide C5a receptor antagonist (PMX205) for 2-3 mo resulted in substantial reduction of pathological markers such as fibrillar amyloid deposits (49-62%) and activated glia (42-68%) in two mouse models of AD. The reduction in pathology was correlated with improvements in a passive avoidance behavioral task in Tg2576 mice. In 3xTg mice, PMX205 also significantly reduced hyperphosphorylated tau (69%). These data provide the first evidence that inhibition of a proinflammatory receptor-mediated function of the complement cascade (i.e., C5aR) can interfere with neuroinflammation and neurodegeneration in AD rodent models, suggesting a novel therapeutic target for reducing pathology and improving cognitive function in human AD patients.