Therapeutic inhibition of the complement system in diseases of the central nervous system

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

Genetic insights into the impact of complement in Alzheimer's disease

The presence of complement activation products at sites of pathology in post-mortem Alzheimer’s disease (AD) brains is well known. Recent evidence from genome-wide association studies (GWAS), combined with the demonstration that complement activation is pivotal in synapse loss in AD, strongly implicates complement in disease aetiology. Genetic variations in complement genes are widespread. While most variants individually have only minor effects on complement homeostasis, the combined effects of variants in multiple complement genes, referred to as the “complotype”, can have major effects. In some diseases, the complotype highlights specific parts of the complement pathway involved in disease, thereby pointing towards a mechanism; however, this is not the case with AD. Here we review the complement GWAS hits; CR1 encoding complement receptor 1 (CR1), CLU encoding clusterin, and a suggestive association of C1S encoding the enzyme C1s, and discuss difficulties in attributing the AD association in these genes to complement function. A better understanding of complement genetics in AD might facilitate predictive genetic screening tests and enable the development of simple diagnostic tools and guide the future use of anti-complement drugs, of which several are currently in development for central nervous system disorders

The complement system in neurodegenerative diseases

Complement is an important component of innate immune defence against pathogens and crucial for efficient immune complex disposal. These core protective activities are dependent in large part on properly regulated complement-mediated inflammation. Dysregulated complement activation, often driven by persistence of activating triggers, is a cause of pathological inflammation in numerous diseases, including neurological diseases. Increasingly, this has become apparent not only in well-recognized neuroinflammatory diseases like multiple sclerosis but also in neurodegenerative and neuropsychiatric diseases where inflammation was previously either ignored or dismissed as a secondary event. There is now a large and rapidly growing body of evidence implicating complement in neurological diseases that cannot be comprehensively addressed in a brief review. Here, we will focus on neurodegenerative diseases, including not only the ‘classical’ neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, but also two other neurological diseases where neurodegeneration is a neglected feature and complement is implicated, namely, schizophrenia, a neurodevelopmental disorder with many mechanistic features of neurodegeneration, and multiple sclerosis, a demyelinating disorder where neurodegeneration is a major cause of progressive decline. We will discuss the evidence implicating complement as a driver of pathology in these diverse diseases and address briefly the potential and pitfalls of anti-complement drug therapy for neurodegenerative diseases

Novel monoclonal antibodies against mouse C1q: characterisation and development of a quantitative ELISA for mouse C1q

Recent studies have identified roles for complement in synaptic pruning, both physiological during development and pathological in Alzheimer’s disease (AD). These reports suggest that C1q initiates complement activation on synapses and C3 fragments then tag them for removal by microglia. There is an urgent need to characterise these processes in rodent AD models; this requires the development of reagents and methods for detection and quantification of rodent C1q in fluids and pathological tissues. These will enable better evaluation of the role of C1q in disease and its value as disease biomarker. We describe the generation in C1q-deficient mice of novel monoclonal antibodies against mouse and rat C1q that enabled development of a sensitive, specific, and quantitative ELISA for mouse and rat C1q capable of measuring C1q in biological fluids and tissue extracts. Serum C1q levels were measured in wild-type (WT), C1q knockout (KO), C3 KO, C7 KO, Crry KO, and 3xTg and APPNL-G-F AD model mice through ageing. C1q levels significantly decreased in WT, APPNL-G-F, and C7 KO mice with ageing. C1q levels were reduced in APPNL-G-F compared to WT at all ages and in 3xTg at 12 months; C3 KO and C7 KO, but not Crry KO mice, also demonstrated significantly lower C1q levels compared to matched WT. In brain homogenates, C1q levels increased with age in both WT and APPNL-G-F mice. This robust and adaptable assay for quantification of mouse and rat C1q provides a vital tool for investigating the expression of C1q in rodent models of AD and other complement-driven pathologies

Quantitative imaging of tissue sections using infrared scanning technology

Rona Barron - ORCID: 0000-0003-4512-9177 https://orcid.org/0000-0003-4512-9177Quantification of immunohistochemically (IHC) labelled tissue sections typically yields semi-quantitative results. Visualising infrared (IR) ‘tags’, with an appropriate scanner, provides an alternative system where the linear nature of the IR fluorophore emittance enables realistic quantitative fluorescence IHC (QFIHC). Importantly, this new technology enables entire tissue sections to be scanned, allowing accurate area and protein abundance measurements to be calculated from rapidly acquired images. Here, some of the potential benefits of using IR-based tissue imaging are examined, and the following are demonstrated. Firstly, image capture and analysis using IR-based scanning technology yields comparable area-based quantification to those obtained from a modern high-resolution digital slide scanner. Secondly, IR-based dual target visualisation and expression-based quantification is rapid and simple. Thirdly, IR-based relative protein abundance QIHC measurements are an accurate reflection of tissue sample protein abundance, as demonstrated by comparison with quantitative fluorescent Western blotting data. In summary, it is proposed that IR-based QFIHC provides an alternative method of rapid whole-tissue section low-resolution imaging for the production of reliable and accurate quantitative data.https://doi.org/10.1111/joa.12398228

Complement receptor 1 is expressed on brain cells and in the human brain

Genome wide association studies (GWAS) have highlighted the importance of the complement cascade in pathogenesis of Alzheimer's disease (AD). Complement receptor 1 (CR1; CD35) is among the top GWAS hits. The long variant of CR1 is associated with increased risk for AD; however, roles of CR1 in brain health and disease are poorly understood. A critical confounder is that brain expression of CR1 is controversial; failure to demonstrate brain expression has provoked the suggestion that peripherally expressed CR1 influences AD risk. We took a multi‐pronged approach to establish whether CR1 is expressed in brain. Expression of CR1 at the protein and mRNA level was assessed in human microglial lines, induced pluripotent stem cell (iPSC)‐derived microglia from two sources and brain tissue from AD and control donors. CR1 protein was detected in microglial lines and iPSC‐derived microglia expressing different CR1 variants when immunostained with a validated panel of CR1‐specific antibodies; cell extracts were positive for CR1 protein and mRNA. CR1 protein was detected in control and AD brains, co‐localizing with astrocytes and microglia, and expression was significantly increased in AD compared to controls. CR1 mRNA expression was detected in all AD and control brain samples tested; expression was significantly increased in AD. The data unequivocally demonstrate that the CR1 transcript and protein are expressed in human microglia ex vivo and on microglia and astrocytes in situ in the human brain; the findings support the hypothesis that CR1 variants affect AD risk by directly impacting glial functions

The impact of complement genes on the risk of late-onset Alzheimer's disease

Late-onset Alzheimer’s disease (LOAD), the most common cause of dementia, and a huge global health challenge, is a neurodegenerative disease of uncertain aetiology. To deliver effective diagnostics and therapeutics, understanding the molecular basis of the disease is essential. Contemporary large genome-wide association studies (GWAS) have identified over seventy novel genetic susceptibility loci for LOAD. Most are implicated in microglial or inflammatory pathways, bringing inflammation to the fore as a candidate pathological pathway. Among the most significant GWAS hits are three complement genes: CLU, encoding the fluid-phase complement inhibitor clusterin; CR1 encoding complement receptor 1 (CR1); and recently, C1S encoding the complement enzyme C1s. Complement activation is a critical driver of inflammation; changes in complement genes may impact risk by altering the inflammatory status in the brain. To assess complement gene association with LOAD risk, we manually created a comprehensive complement gene list and tested these in gene-set analysis with LOAD summary statistics. We confirmed associations of CLU and CR1 genes with LOAD but showed no significant associations for the complement gene-set when excluding CLU and CR1. No significant association with other complement genes, including C1S, was seen in the IGAP dataset; however, these may emerge from larger datasets

Terminal complement pathway activation drives synaptic Loss in Alzheimer’s disease models

Complement is involved in developmental synaptic pruning and pathological synapse loss in Alzheimer’s disease. It is posited that C1 binding initiates complement activation on synapses; C3 fragments then tag them for microglial phagocytosis. However, the precise mechanisms of complement-mediated synaptic loss remain unclear, and the role of the lytic membrane attack complex (MAC) is unexplored. We here address several knowledge gaps: (i) is complement activated through to MAC at the synapse? (ii) does MAC contribute to synaptic loss? (iii) can MAC inhibition prevent synaptic loss? Novel methods were developed and optimised to quantify C1q, C3 fragments and MAC in total and regional brain homogenates and synaptoneurosomes from WT and AppNL−G−F Alzheimer’s disease model mouse brains at 3, 6, 9 and 12 months of age. The impact on synapse loss of systemic treatment with a MAC blocking antibody and gene knockout of a MAC component was assessed in Alzheimer’s disease model mice. A significant increase in C1q, C3 fragments and MAC was observed in AppNL−G−F mice compared to controls, increasing with age and severity. Administration of anti-C7 antibody to AppNL−G−F mice modulated synapse loss, reflected by the density of dendritic spines in the vicinity of plaques. Constitutive knockout of C6 significantly reduced synapse loss in 3xTg-AD mice. We demonstrate that complement dysregulation occurs in Alzheimer’s disease mice involving the activation (C1q; C3b/iC3b) and terminal (MAC) pathways in brain areas associated with pathology. Inhibition or ablation of MAC formation reduced synapse loss in two Alzheimer’s disease mouse models, demonstrating that MAC formation is a driver of synapse loss. We suggest that MAC directly damages synapses, analogous to neuromuscular junction destruction in myasthenia gravis

Analysis of gene expression in the nervous system identifies key genes and novel candidates for health and disease

The incidence of neurodegenerative diseases in the developed world has risen over the last century, concomitant with an increase in average human lifespan. A major challenge is therefore to identify genes that control neuronal health and viability with a view to enhancing neuronal health during ageing and reducing the burden of neurodegeneration. Analysis of gene expression data has recently been used to infer gene functions for a range of tissues from co-expression networks. We have now applied this approach to transcriptomic datasets from the mammalian nervous system available in the public domain. We have defined the genes critical for influencing neuronal health and disease in different neurological cell types and brain regions. The functional contribution of genes in each co-expression cluster was validated using human disease and knockout mouse phenotypes, pathways and gene ontology term annotation. Additionally a number of poorly annotated genes were implicated by this approach in nervous system function. Exploiting gene expression data available in the public domain allowed us to validate key nervous system genes and, importantly, to identify additional genes with minimal functional annotation but with the same expression pattern. These genes are thus novel candidates for a role in neurological health and disease and could now be further investigated to confirm their function and regulation during ageing and neurodegeneration. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10048-017-0509-5) contains supplementary material, which is available to authorized users