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.

Complement as a powerful "influencer" in the brain during development, adulthood and neurological disorders.

The complement system was long considered as only a powerful effector arm of the immune system that, while critically protective, could lead to inflammation and cell death if overactivated, even in the central nervous system (CNS). However, in the past decade it has been recognized as playing critical roles in key physiological processes in the CNS, including neurogenesis and synaptic remodeling in the developing and adult brain. Inherent in these processes are the interactions with cells in the brain, and the cascade of interactions and functional consequences that ensue. As a result, investigations of therapeutic approaches for both suppressing excessive complement driven neurotoxicity and aberrant sculpting of neuronal circuits, require broad (and deep) knowledge of the functional activities of multiple components of this highly evolved and regulated system to avoid unintended negative consequences in the clinic. Advances in basic science are beginning to provide a roadmap for translation to therapeutics, with both small molecule and biologics. Here, we present examples of the critical roles of proper complement function in the development and sculpting of the nervous system, and in enabling rapid protection from infection and clearance of dying cells. Microglia are highlighted as important command centers that integrate signals from the complement system and other innate sensors that are programed to provide support and protection, but that direct detrimental responses to aberrant activation and/or regulation of the system. Finally, we present promising research areas that may lead to effective and precision strategies for complement targeted interventions to promote neurological health.

Peripheral viral challenge increases c-fos level in cerebral neurons.

Peripheral viral infection can substantially alter brain function. We have previously shown that intraperitoneal (i.p.) injection of a viral mimetic, polyinosinic-polycytidylic acid (PIC), engenders hyperexcitability of cerebral neurons. Because neuronal activity is invariably associated with their expression of the Cfos gene, the present study was undertaken to determine whether PIC challenge also increases neuronal c-fos protein level. Female C57BL/6 mice were i.p. injected with PIC, and neuronal c-fos was analyzed in the motor cortex by immunohistochemistry. PIC challenge instigated a robust increase in the number of c-fos-positive neurons. This increase reached approximately tenfold over control at 24 h. Also, the c-fos staining intensity of individual neurons increased. AMG-487, a specific inhibitor of the chemokine receptor CXCR3, profoundly attenuated the accumulation of neuronal c-fos, indicating the activation of CXCL10/CXCR3 axis as the trigger of the process. Together, these results show that the accumulation of c-fos is a viable readout to assess the response of cerebral neurons to peripheral PIC challenge, and to elucidate the underlying molecular mechanisms.

Neuronal CXCL10/CXCR3 Axis Mediates the Induction of Cerebral Hyperexcitability by Peripheral Viral Challenge.

Peripheral infections can potently exacerbate neuropathological conditions, though the underlying mechanisms are poorly understood. We have previously demonstrated that intraperitoneal (i.p.) injection of a viral mimetic, polyinosinic-polycytidylic acid (PIC) induces a robust generation of CXCL10 chemokine in the hippocampus. The hippocampus also features hyperexcitability of neuronal circuits following PIC challenge. The present study was undertaken to determine the role of CXCL10 in mediating the development of hyperexcitability in response to PIC challenge. Briefly, young female C57BL/6 mice were i.p. injected with PIC, and after 24 h, the brains were analyzed by confocal microscopy. CXCL10 staining of neuronal perikarya and a less intense staining of the neuropil was observed in the hippocampus and cortex. CXCL10 staining was also evident in a subpopulation of astrocytes, whereas microglia were CXCL10 negative. CXCR3, the cognate receptor of CXCL10 was present exclusively on neurons, indicating that the CXCL10/CXCR3 axis operates through an autocrine/paracrine neuronal signaling. Blocking cerebral CXCR3 through intracerebroventricular injection of a specific inhibitor, AMG487, abrogated PIC challenge-induced increase in basal synaptic transmission and long-term potentiation (LTP), as well as the reduction of paired-pulse facilitation (PPF), in the hippocampus. The PIC-mediated abolishment of hippocampal long-term depression (LTD) was also restored after administration of AMG487. Moreover, CXCR3 inhibition attenuated seizure hypersensitivity induced by PIC challenge. The efficacy of AMG487 strongly strengthens the notion that CXCL10/CXCR3 axis mediates the induction of cerebral hyperexcitability by PIC challenge.

Peripheral viral challenge exacerbates experimental autoimmune encephalomyelitis.

Peripheral viral infections are potent triggers of exacerbation in multiple sclerosis (MS). Here, we used a preclinical model of MS, the experimental autoimmune encephalomyelitis (EAE) to corroborate this comorbidity in an experimental setting. EAE was induced by immunization of mice with MOG peptide, and paralysis was scored using a 5-point scale. At the onset of the chronic phase of the disease (Days 42-58 after MOG injection) the animals were divided into low responders (LR) and high responders (HR) with the mean score of 1.5 and 2.5, respectively. The acute phase response (APR) was induced by intraperitoneal injections of a viral mimetic, polyinosinic-polycytidylic acid (PIC). Two daily injections were performed on Days 42 and 44 (PIC42,44 challenge) and on Days 54, 55 and 56 (PIC54,55,56 challenge). PIC42,44 challenge had no effect of EAE disease, whereas PIC54,55,56 challenge rapidly increased paralysis but only in HR group. This exacerbation ultimately led to animal death by Day 58. These results demonstrate that antiviral APR is a potent exacerbator of EAE, and that this activity directly correlates with the severity of the disease. This in turn, indicates that antiviral APR might play a pivot role in linking peripheral viral infections with MS exacerbations.

Peripherally restricted viral challenge elevates extracellular glutamate and enhances synaptic transmission in the hippocampus.

Peripheral infections increase the propensity and severity of seizures in susceptible populations. We have previously shown that intraperitoneal injection of a viral mimic, polyinosinic-polycytidylic acid (PIC), elicits hypersusceptibility of mice to kainic acid (KA)-induced seizures. This study was undertaken to determine whether this seizure hypersusceptibility entails alterations in glutamate signaling. Female C57BL/6 mice were intraperitoneally injected with PIC, and after 24 h, glutamate homeostasis in the hippocampus was monitored using the enzyme-based microelectrode arrays. PIC challenge robustly increased the level of resting extracellular glutamate. While pre-synaptic potassium-evoked glutamate release was not affected, glutamate uptake was profoundly impaired and non-vesicular glutamate release was augmented, indicating functional alterations of astrocytes. Electrophysiological examination of hippocampal slices from PIC-challenged mice revealed a several fold increase in the basal synaptic transmission as compared to control slices. PIC challenge also increased the probability of pre-synaptic glutamate release as seen from a reduction of paired-pulse facilitation and synaptic plasticity as seen from an enhancement of long-term potentiation. Altogether, our results implicate a dysregulation of astrocytic glutamate metabolism and an alteration of excitatory synaptic transmission as the underlying mechanism for the development of hippocampal hyperexcitability, and consequently seizure hypersusceptibility following peripheral PIC challenge. Peripheral infections/inflammations enhance seizure susceptibility. Here, we explored the effect of peritoneal inflammation induced by a viral mimic on glutamate homeostasis and glutamatergic neurotransmission in the mouse hippocampus. We found that peritoneal inflammation elevated extracellular glutamate concentration and enhanced the probability of pre-synaptic glutamate release resulting in hyperexcitability of neuronal networks. These mechanisms are likely to underlie the enhanced seizure propensity.