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The complement system plays critical roles in development, homeostasis, and regeneration in the central nervous system (CNS) throughout life; however, complement dysregulation in the CNS can lead to damage and disease. Complement proteins, regulators, and receptors are widely expressed throughout the CNS and, in many cases, are upregulated in disease. Genetic and epidemiological studies, cerebrospinal fluid (CSF) and plasma biomarker measurements and pathological analysis of post-mortem tissues have all implicated complement in multiple CNS diseases including multiple sclerosis (MS), neuromyelitis optica (NMO), neurotrauma, stroke, amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Given this body of evidence implicating complement in diverse brain diseases, manipulating complement in the brain is an attractive prospect; however, the blood-brain barrier (BBB), critical to protect the brain from potentially harmful agents in the circulation, is also impermeable to current complement-targeting therapeutics, making drug design much more challenging. For example, antibody therapeutics administered systemically are essentially excluded from the brain. Recent protocols have utilized \"Trojan horse\" techniques to transport therapeutics across the BBB or used osmotic shock or ultrasound to temporarily disrupt the BBB. Most research to date exploring the impact of complement inhibition on CNS diseases has been in animal models, and some of these studies have generated convincing data; for example, in models of MS, NMO, and stroke. There have been a few recent clinical trials of available anti-complement drugs in CNS diseases associated with BBB impairment, for example the use of the anti-C5 monoclonal antibody (mAb) eculizumab in NMO, but for most CNS diseases there have been no human trials of anti-complement therapies. Here we will review the evidence implicating complement in diverse CNS disorders, from acute, such as traumatic brain or spine injury, to chronic, including demyelinating, neuroinflammatory, and neurodegenerative diseases. We will discuss the particular problems of drug access into the CNS and explore ways in which anti-complement therapies might be tailored for CNS disease.

Dendritic spines are the postsynaptic sites for most excitatory glutamatergic synapses. We previously demonstrated a severe spine loss and synaptic reorganization in human neocortices presenting Type II focal cortical dysplasia (FCD), a developmental malformation and frequent cause of drug‐resistant focal epilepsy. We extend the findings, investigating the potential role of complement components C1q and C3 in synaptic pruning imbalance. Data from Type II FCD were compared with those obtained in focal epilepsies with different etiologies. Neocortical tissues were collected from 20 subjects, mainly adults with a mean age at surgery of 31 years, admitted to epilepsy surgery with a neuropathological diagnosis of: cryptogenic, temporal lobe epilepsy with hippocampal sclerosis, and Type IIa/b FCD. Dendritic spine density quantitation, evaluated in a previous paper using Golgi impregnation, was available in a subgroup. Immunohistochemistry, in situ hybridization, electron microscopy, and organotypic cultures were utilized to study complement/microglial activation patterns. FCD Type II samples presenting dendritic spine loss were characterized by an activation of the classical complement pathway and microglial reactivity. In the same samples, a close relationship between microglial cells and dendritic segments/synapses was found. These features were consistently observed in Type IIb FCD and in 1 of 3 Type IIa cases. In other patient groups and in perilesional areas outside the dysplasia, not presenting spine loss, these features were not observed. In vitro treatment with complement proteins of organotypic slices of cortical tissue with no sign of FCD induced a reduction in dendritic spine density. These data suggest that dysregulation of the complement system plays a role in microglia‐mediated spine loss. This mechanism, known to be involved in the removal of redundant synapses during development, is likely reactivated in Type II FCD, particularly in Type IIb; local treatment with anticomplement drugs could in principle modify the course of disease in these patients.

Background Alzheimer’s disease (AD) has been associated with immune dysregulation in biomarker and genome-wide association studies (GWAS). GWAS hits include the genes encoding complement regulators clusterin ( CLU ) and complement receptor 1 ( CR1 ), recognised as key players in AD pathology, and complement proteins have been proposed as biomarkers. Main body To address whether changes in plasma complement protein levels in AD relate to AD-associated complement gene variants we first measured relevant plasma complement proteins (clusterin, C1q, C1s, CR1, factor H) in a large cohort comprising early onset AD (EOAD; n = 912), late onset AD (LOAD; n = 492) and control (n = 504) donors. Clusterin and C1q were significantly increased (p < 0.001) and sCR1 and factor H reduced (p < 0.01) in AD plasma versus controls. ROC analyses were performed to assess utility of the measured complement biomarkers, alone or in combination with amyloid beta, in predicting AD. C1q was the most predictive single complement biomarker (AUC 0.655 LOAD, 0.601 EOAD); combining C1q with other complement or neurodegeneration makers through stepAIC-informed models improved predictive values slightly. Effects of GWS SNPs (rs6656401, rs6691117 in CR1 ; rs11136000, rs9331888 in CLU ; rs3919533 in C1S ) on protein concentrations were assessed by comparing protein levels in carriers of the minor vs major allele. To identify new associations between SNPs and changes in plasma protein levels, we performed a GWAS combining genotyping data in the cohort with complement protein levels as endophenotype. SNPs in CR1 (rs6656401), C1S (rs3919533) and CFH (rs6664877) reached significance and influenced plasma levels of the corresponding protein, whereas SNPs in CLU did not influence clusterin levels. Conclusion Complement dysregulation is evident in AD and may contribute to pathology. AD-associated SNPs in CR1 , C1S and CFH impact plasma levels of the encoded proteins, suggesting a mechanism for impact on disease risk.

C4d deposition in peritubular capillaries (PTC) reflects complement activation in antibody-mediated rejection (ABMR) of kidney allograft. However, its association with allograft survival is controversial. We hypothesized that capillary deposition of C5b9-indicative of complement-mediated injury-is a severity marker of ABMR. This pilot study aimed to determine the frequency, location and prognostic impact of these deposits in ABMR. We retrospectively selected patients diagnosed with ABMR in two French transplantation centers from January 2005 to December 2014 and performed C4d and C5b9 staining by immunohistochemistry. Fifty-four patients were included. Median follow-up was 52.5 (34.25-73.5) months. Thirteen patients (24%) had C5b9 deposits along glomerular capillaries (GC). Among these, seven (54%) had a global and diffuse staining pattern. Twelve of the C5b9+ patients also had deposition of C4d in GC and PTC. C4d deposits along GC and PTC were not associated with death-censored allograft survival ( = 0.42 and 0.69, respectively). However, death-censored allograft survival was significantly lower in patients with global and diffuse deposition of C5b9 in GC than those with a segmental pattern or no deposition (median survival after ABMR diagnosis, 6 months, 40.5 months and 44 months, respectively; = 0.015). Double contour of glomerular basement membrane was diagnosed earlier after transplantation in C5b9+ ABMR than in C5b9- ABMR (median time after transplantation, 28 vs. 85 months; = 0.058). In conclusion, we identified a new pattern of C5b9+ ABMR, associated with early onset of glomerular basement membrane duplication and poor allograft survival. Complement inhibitors might be a therapeutic option for this subgroup of patients.

INTRODUCTION Genome‐wide association studies have implicated complement in Alzheimer's disease (AD). The CR1*2 variant of complement receptor 1 (CR1; CD35), confers increased AD risk. We confirmed CR1 expression on glial cells; however, how CR1 variants influence AD risk remains unclear. METHODS Induced pluripotent stem cell‐derived microglia and astrocytes were generated from donors homozygous for the common CR1 variants (CR1*1/CR1*1;CR1*2/CR1*2). CR1 expression was quantified and phagocytic activity assessed using diverse targets (Escherichia coli bioparticles, amyloid β aggregates, and synaptoneurosomes), with or without serum opsonization. RESULTS Expression of CR1*1 was significantly higher than CR1*2 on glial lines. Phagocytosis for all targets was markedly enhanced following serum opsonization, attenuated by Factor I‐depletion, demonstrating CR1 requirement for C3b processing. CR1*2‐expressing glia showed significantly enhanced phagocytosis of all opsonized targets compared to CR1*1‐expressing cells. DISCUSSION CR1 is critical for glial phagocytosis of opsonized targets. CR1*2, despite lower expression, enhances glial phagocytosis, providing mechanistic explanation of increased AD risk. Highlights Induced pluripotent stem cell (iPSC)‐derived glia from individuals expressing the Alzheimer's disease (AD) risk variant complement receptor (CR) 1*2 exhibit lower CR1 expression compared to those from donors expressing the non‐risk form CR1*1. The iPSC‐derived glia from individuals expressing the AD risk variant CR1*2 exhibit enhanced phagocytic activity for opsonized bacterial particles, amyloid‐β aggregates and human synaptoneurosomes compared to those from donors expressing the non‐risk form CR1*1. We suggest that expression of the CR1*2 variant confers risk of AD by enhancing the phagocytic capacity of glia for opsonized targets.  

Background Neuroinflammation is a critical factor of Alzheimer’s Disease (AD). Dysregulation of complement leads to excessive inflammation, direct damage to self‐cells and propagation of injury. This is likely of particular relevance in the brain where inflammation is poorly tolerated and brain cells are vulnerable to direct damage by complement. Membrane attack complex (MAC) is highly pro‐inflammatory product of the complement cascade killing cells by lysis and/or causing ‘bystander’ damage initiating NLRP3 inflammasome activation and provoking other immune damaging responses leading to death of the vulnerable nerve cells. Method The role of MAC in AD was investigated in MAC‐deficient animals and by using a newly developed anti‐C7 monoclonal antibody (mAb) that efficiently inhibits formation of the MAC in vitro and in vivo. Impact of C7 deficiency on brain complement dysregulation, synapse loss, amyloid load and cognitive decline was examined by comparing APPNL‐G‐F mice back‐crossed to C7 deficiency (APPNL‐G‐FxC7) with unmodified APPNL‐G‐F mice. To assess the effect of therapeutic C7 blockade, unmodified APPNL‐G‐F mice were treated systemically (for four weeks) with anti‐C7 mAb or control IgG and the same set of parameters of complement dysregulation, pathology and cognition measured. Result C7 deficiency in AppNL−G−F mice reduced levels of complement activation markers, reduced amyloid load and increased synapse density with a commensurate improvement in cognitive test performance. Systemic treatment of AppNL−G−F mice with a blocking anti‐C7 mAb reduced brain levels of complement activation markers, amyloid load and increased neuronal spine density in treated mice in peri‐plaque areas when compared to controls. in AppNL−G−F mice. APPNL‐G‐FxC7 performed significanlty better in behavioural cognitive tests. Conclusion We demonstrate that complement dysregulation occurs in brain in mouse models of AD. C7 deficiency reduced brain complement dysregulation, improved pathological parameters and cognitive function; systemic anti‐C7 therapy reduced complement dysregulation and protected from synapse loss in the model. Modification for brain delivery of the anti‐C7 mAb will enhance efficacy in the model. The findings highlight the potential for complement inhibition at the level of MAC as a therapy in AD.

There is a growing body of evidence implicating complement and, in particular, the terminal pathway (membrane attack complex; MAC) in inducing demyelination in multiple sclerosis and experimental allergic encephalomyelitis. In this paper, we examined the disease course and pathological changes in mice deficient in the major regulator of MAC assembly, CD59a, during the course of acute experimental allergic encephalomyelitis induced by immunisation with recombinant myelin oligodendrocyte glycoprotein. Disease incidence and severity were significantly increased in CD59a-deficient mice. The extent of inflammation, demyelination and axonal injury were assessed in spinal cord cross-sections from CD59a-deficient and control mice, and all these parameters were enhanced in the absence of CD59a. Areas of myelin loss and axonal damage in CD59a-deficient mice were associated with deposits of MAC, firmly implicating MAC as a cause of the observed injury. These findings are relevant to some types of human demyelination, where abundant deposits of MAC are found in association with pathology.

Background Complement contributes significantly to pathophysiology in many neurodegenerative diseases (NDDs) including Alzheimer's Disease (AD); yet, the source of complement in the brain is poorly understood. Even multiple small changes to individual complement components can compound into significant dysregulation of the whole system. Targeting complement effectively in NDDs requires understanding complement expression in the healthy brain and how it changes in pathology. Method We integrated single‐nucleus RNA sequencing datasets, including over 600,000 cells from 97 donor frontal cortices, 60 AD donors (36 male, 24 female) and 37 control donors (23 male, 14 female). Complement expression across nine cell types, and the impact of AD pathology, sex and age on complement gene expression was examined. Results Microglia are the principle source of C1Q/A/B/C and C3 expression in both the healthy and AD brain, with more microglia expressing higher levels observed in AD. Increases in C1R, C1S, C5 and C7 expression are seen in AD, predominately in fibroblasts, pericytes and astrocytes. Endothelial cells strongly expressed most complement regulators including, CD46, CD55, CD59 and CFH with greater expression observed in AD. The AD risk gene CLU demonstrated significantly higher expression in multiple cell types however, remains predominantly expressed in astrocytes. Expression of the novel complement regulator SRPX2 was markedly increased in AD in pericytes and fibroblasts, while slightly elevated expression of CSMD1/2/3 is seen in neurons. No significant differences in complement expression between sexes was observed in control donors. However, in AD donors sex‐based differences were seen with female donors typically demonstrating a greater percentage of cells expressing complement. This data suggests that AD alters the normal pattern of changes in complement gene expression with age, in particular, significantly higher expression of C1R, C1S and CLU are seen in the youngest age group. Conclusion Our comprehensive complement expression atlas for the AD and control frontal cortex reveals profound complement dysregulation in AD. There are sex specific trends in this dysregulation with females demonstrating greater dysregulation. Finally, most severe complement dysregulation in AD is observed in the youngest individuals highlighting a significant age‐dependent factor that could inform the timing and design of therapeutic interventions.

Complement contributes significantly to pathophysiology in many neurodegenerative diseases (NDDs) including Alzheimer's Disease (AD); yet, the source of complement in the brain is poorly understood. Even multiple small changes to individual complement components can compound into significant dysregulation of the whole system. Targeting complement effectively in NDDs requires understanding complement expression in the healthy brain and how it changes in pathology. We integrated single-nucleus RNA sequencing datasets, including over 600,000 cells from 97 donor frontal cortices, 60 AD donors (36 male, 24 female) and 37 control donors (23 male, 14 female). Complement expression across nine cell types, and the impact of AD pathology, sex and age on complement gene expression was examined. Microglia are the principle source of C1Q/A/B/C and C3 expression in both the healthy and AD brain, with more microglia expressing higher levels observed in AD. Increases in C1R, C1S, C5 and C7 expression are seen in AD, predominately in fibroblasts, pericytes and astrocytes. Endothelial cells strongly expressed most complement regulators including, CD46, CD55, CD59 and CFH with greater expression observed in AD. The AD risk gene CLU demonstrated significantly higher expression in multiple cell types however, remains predominantly expressed in astrocytes. Expression of the novel complement regulator SRPX2 was markedly increased in AD in pericytes and fibroblasts, while slightly elevated expression of CSMD1/2/3 is seen in neurons. No significant differences in complement expression between sexes was observed in control donors. However, in AD donors sex-based differences were seen with female donors typically demonstrating a greater percentage of cells expressing complement. This data suggests that AD alters the normal pattern of changes in complement gene expression with age, in particular, significantly higher expression of C1R, C1S and CLU are seen in the youngest age group. Our comprehensive complement expression atlas for the AD and control frontal cortex reveals profound complement dysregulation in AD. There are sex specific trends in this dysregulation with females demonstrating greater dysregulation. Finally, most severe complement dysregulation in AD is observed in the youngest individuals highlighting a significant age-dependent factor that could inform the timing and design of therapeutic interventions.

Background In the brain as in other organs, complement contributes to immune defence and housekeeping to maintain homeostasis. Sources of complement may include local production by brain cells and influx from the periphery, the latter severely restricted by the blood brain barrier (BBB) in healthy brain. Dysregulation of complement leads to excessive inflammation, direct damage to self‐cells and propagation of injury. This is likely of particular relevance in the brain where inflammation is poorly tolerated and brain cells are vulnerable to direct damage by complement. Method We have developed novel anti‐C7 antibodies (mAb) that efficiently inhibit formation of the pro‐inflammatory membrane attack complex (MAC) in vitro and in vivo. Here we describe recombinant fusion proteins (FP) that replicate the MAC‐blocking action of the mAb, and are designed to access the brain utilising “Trojan horse” shuttles. The Alzheimer model APPNL‐G‐F mice were treated systemically with native mAb to swamp peripheral C7 followed by the FP. Immunohistochemistry and ELISA were used to demonstrate FP entry into brain and show impact on the disease pathology. Result The recombinant FP showed complement inhibitory activity in vitro equivalent to their parent mAb and were able to cross an artificial BBB in transwells. The presence of the FP in brain homogenates of peripherally dosed animals was confirmed by ELISA. Treatment with the FP caused reduced levels of complement activation products C3b and terminal complement complex (TCC) in brain. Diolistics analysis showed significant increased neuronal spine density in treated mice compared to controls, demonstrating a protective effect of the FP on synaptic function. Mice treated with the drug showed significant improvements in cognition. Conclusion The FP described are able to cross BBB and are potent inhibitors of complement in brain; impact on brain pathology was detected after just one week of treatment. The findings highlight the potential for complement inhibition as a therapy in Alzheimer’s disease.