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.

Alzheimer's disease-associated complement gene variants influence plasma complement protein levels.

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.

Ca2+ Influx through TRPC Channels Is Regulated by Homocysteine-Copper Complexes.

An elevated level of circulating homocysteine (Hcy) has been regarded as an independent risk factor for cardiovascular disease; however, the clinical benefit of Hcy lowering-therapy is not satisfying. To explore potential unrevealed mechanisms, we investigated the roles of Ca2+ influx through TRPC channels and regulation by Hcy-copper complexes. Using primary cultured human aortic endothelial cells and HEK-293 T-REx cells with inducible TRPC gene expression, we found that Hcy increased the Ca2+ influx in vascular endothelial cells through the activation of TRPC4 and TRPC5. The activity of TRPC4 and TRPC5 was regulated by extracellular divalent copper (Cu2+) and Hcy. Hcy prevented channel activation by divalent copper, but monovalent copper (Cu+) had no effect on the TRPC channels. The glutamic acids (E542/E543) and the cysteine residue (C554) in the extracellular pore region of the TRPC4 channel mediated the effect of Hcy-copper complexes. The interaction of Hcy-copper significantly regulated endothelial proliferation, migration, and angiogenesis. Our results suggest that Hcy-copper complexes function as a new pair of endogenous regulators for TRPC channel activity. This finding gives a new understanding of the pathogenesis of hyperhomocysteinemia and may explain the unsatisfying clinical outcome of Hcy-lowering therapy and the potential benefit of copper-chelating therapy.

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.

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.

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.

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.

ORAI channels are critical for receptor-mediated endocytosis of albumin.

Impaired albumin reabsorption by proximal tubular epithelial cells (PTECs) has been highlighted in diabetic nephropathy (DN), but little is known about the underlying molecular mechanisms. Here we find that ORAI1-3, are preferentially expressed in PTECs and downregulated in patients with DN. Hyperglycemia or blockade of insulin signaling reduces the expression of ORAI1-3. Inhibition of ORAI channels by BTP2 and diethylstilbestrol or silencing of ORAI expression impairs albumin uptake. Transgenic mice expressing a dominant-negative Orai1 mutant (E108Q) increases albuminuria, and in vivo injection of BTP2 exacerbates albuminuria in streptozotocin-induced and Akita diabetic mice. The albumin endocytosis is Ca2+-dependent and accompanied by ORAI1 internalization. Amnionless (AMN) associates with ORAIs and forms STIM/ORAI/AMN complexes after Ca2+ store depletion. STIM1/ORAI1 colocalizes with clathrin, but not with caveolin, at the apical membrane of PTECs, which determines clathrin-mediated endocytosis. These findings provide insights into the mechanisms of protein reabsorption and potential targets for treating diabetic proteinuria.

COL1A1, PRPF40A, and UCP2 correlate with hypoxia markers in non-small cell lung cancer.

PURPOSE: Collagen 1A1 (COL1A1), RNA-binding and pre-mRNA Processing Factor (PRPF40A), and Uncoupling Protein 2 (UCP2) were identified as downstream effectors of cytoglobin (CYGB), which was shown implicated in tumour biology. Although these three genes have been previously associated with cancer, little is known about their status in lung malignancies. METHODS: Hereby, we investigated the expression and promoter methylation of COL1A1, PRPF40A, and UCP2 in 156 non-small cell lung cancer (NSCLC) and adjacent normal tissues. RESULTS: We demonstrate that COL1A1 and PRPF40A mRNAs are significantly overexpressed in NSCLC (p < 1 × 10-4), while UCP2 exhibits a trend of upregulation (p = 0.066). Only COL1A1 promoter revealed hypermethylation in NSCLCs (36%), which was particularly evident in squamous cell carcinomas (p = 0.024) and in the tumours with moderate-to-good differentiation (p = 0.01). Transcript level of COL1A1, as well as PRPF40A and UCP2, exhibited striking association (p ≤ 0.001) with the expression of hypoxia markers. In addition, we demonstrate in lung cancer cell lines exposed to hypoxia or oxidative stress that COL1A1 transcription significantly responds to oxygen depletion, while other genes showed the modest upregulation in stress conditions. CONCLUSION: In conclusion, our data revealed that COL1A1, UCP2, and PRPF40A are novel players implicated in the complex network of hypoxia response in NSCLC.

High glucose enhances store-operated calcium entry by upregulating ORAI/STIM via calcineurin-NFAT signalling.

UNLABELLED: ORAI and stromal interaction molecule (STIM) are store-operated channel molecules that play essential roles in human physiology through a coupling mechanism of internal Ca(2+) store to Ca(2+) influx. However, the roles of ORAI and STIM in vascular endothelial cells under diabetic conditions remain unknown. Here, we investigated expression and signalling pathways of ORAI and STIM regulated by high glucose or hyperglycaemia using in vitro cell models, in vivo diabetic mice and tissues from patients. We found that ORAI1-3 and STIM1-2 were ubiquitously expressed in human vasculatures. Their expression was upregulated by chronic treatment with high glucose (HG, 25 mM D-glucose), which was accompanied by enhanced store-operated Ca(2+) influx in vascular endothelial cells. The increased expression was also observed in the aortae from genetically modified Akita diabetic mice (C57BL/6-Ins2(Akita)/J) and streptozocin-induced diabetic mice, and aortae from diabetic patients. HG-induced upregulation of ORAI and STIM genes was prevented by the calcineurin inhibitor cyclosporin A and NFATc3 siRNA. Additionally, in vivo treatment with the nuclear factor of activated T cells (NFAT) inhibitor A-285222 prevented the gene upregulation in Akita mice. However, HG had no direct effects on ORAI1-3 currents and the channel activation process through cytosolic STIM1 movement in the cells co-expressing STIM1-EYFP/ORAIs. We concluded that upregulation of STIM/ORAI through Ca(2+)-calcineurin-NFAT pathway is a novel mechanism causing abnormal Ca(2+) homeostasis and endothelial dysfunction under hyperglycaemia. KEY MESSAGE: ORAI1-3 and STIM1-2 are ubiquitously expressed in vasculatures and upregulated by high glucose. Increased expression is confirmed in Akita (Ins2(Akita)/J) and STZ diabetic mice and patients. Upregulation mechanism is mediated by Ca(2+)/calcineurin/NFATc3 signalling. High glucose has no direct effects on ORAI1-3 channel activity and channel activation process.

The ryanodine receptor agonist 4-chloro-3-ethylphenol blocks ORAI store-operated channels.

BACKGROUND: Depletion of the Ca(2+) store by ryanodine receptor (RyR) agonists induces store-operated Ca(2+) entry (SOCE). 4-Chloro-3-ethylphenol (4-CEP) and 4-chloro-m-cresol (4-CmC) are RyR agonists commonly used as research tools and diagnostic reagents for malignant hyperthermia. Here, we investigated the effects of 4-CEP and its analogues on SOCE. EXPERIMENTAL APPROACH: SOCE and ORAI1-3 currents were recorded by Ca(2+) imaging and whole-cell patch recordings in rat L6 myoblasts and in HEK293 cells overexpressing STIM1/ORAI1-3. KEY RESULTS: 4-CEP induced a significant release of Ca(2+) in rat L6 myoblasts, but inhibited SOCE. The inhibitory effect was concentration-dependent and more potent than its analogues 4-CmC and 4-chlorophenol (4-ClP). In the HEK293 T-REx cells overexpressing STIM1/ORAI1-3, 4-CEP inhibited the ORAI1, ORAI2 and ORAI3 currents evoked by thapsigargin. The 2-APB-induced ORAI3 current was also blocked by 4-CEP. This inhibitory effect was reversible and independent of the Ca(2+) release. The two analogues, 4-CmC and 4-ClP, also inhibited the ORAI1-3 channels. Excised patch and intracellular application of 4-CEP demonstrated that the action site was located extracellularly. Moreover, 4-CEP evoked STIM1 translocation and subplasmalemmal clustering through its Ca(2+) store-depleting effect via the activation of RyR, but no effect on STIM1 redistribution was observed in cells co-expressing STIM1/ORAI1-3. CONCLUSION AND IMPLICATIONS: 4-CEP not only acts as a RyR agonist to deplete the Ca(2+) store and trigger STIM1 subplasmalemmal translocation and clustering, but also directly inhibits ORAI1-3 channels. These findings demonstrate a novel pharmacological property for the chlorophenol derivatives that act as RyR agonists.

Involvement of TRPC channels in lung cancer cell differentiation and the correlation analysis in human non-small cell lung cancer.

The canonical transient receptor potential (TRPC) channels are Ca(2+)-permeable cationic channels controlling the Ca(2+) influx evoked by G protein-coupled receptor activation and/or by Ca(2+) store depletion. Here we investigate the involvement of TRPCs in the cell differentiation of lung cancer. The expression of TRPCs and the correlation to cancer differentiation grade in non-small cell lung cancer (NSCLC) were analyzed by real-time PCR and immunostaining using tissue microarrays from 28 patient lung cancer samples. The association of TRPCs with cell differentiation was also investigated in the lung cancer cell line A549 by PCR and Western blotting. The channel activity was monitored by Ca(2+) imaging and patch recording after treatment with all-trans-retinoic acid (ATRA). The expression of TRPC1, 3, 4 and 6 was correlated to the differentiation grade of NSCLC in patients, but there was no correlation to age, sex, smoking history and lung cancer cell type. ATRA upregulated TRPC3, TRPC4 and TRPC6 expression and enhanced Ca(2+) influx in A549 cells, however, ATRA showed no direct effect on TRPC channels. Inhibition of TRPC channels by pore-blocking antibodies decreased the cell mitosis, which was counteracted by chronic treatment with ATRA. Blockade of TRPC channels inhibited A549 cell proliferation, while overexpression of TRPCs increased the proliferation. We conclude that TRPC expression correlates to lung cancer differentiation. TRPCs mediate the pharmacological effect of ATRA and play important roles in regulating lung cancer cell differentiation and proliferation, which gives a new understanding of lung cancer biology and potential anti-cancer therapy.

Cytoglobin has bimodal: tumour suppressor and oncogene functions in lung cancer cell lines.

Cytoglobin (CYGB) is frequently downregulated in many types of human malignancies, and its exogenous overexpression reduces proliferation of cancer cells. Despite its implied tumour suppressor (TSG) functions, its exact role in carcinogenesis remains unclear as CYGB upregulation is also associated with tumour hypoxia and aggressiveness. In this study, we explore the TSG role of CYGB, its influence on the phenotype of cancerous cells under stress conditions and the clinical significance of CYGB expression and promoter methylation in non-small cell lung cancer (NSCLC). DNA methylation-dependent expression silencing of CYGB is demonstrated in both clinical samples and cell lines. CYGB promoter was more frequently methylated in lung adenocarcinomas (P = 1.4 × 10(-4)). Demethylation by 5'-azadeoxycytidine partially restored CYGB expression in cell lines. Interestingly, trichostatin A triggered upregulation of CYGB expression in cancer cell lines and downregulation in non-tumourigenic ones. CYGB mRNA expression in NSCLC surgical specimens correlated with that of HIF1α and VEGFa (P < 1 × 10(-4)). Overexpression of CYGB in cancer cell lines reduced cell migration, invasion and anchorage-independent growth. Moreover, CYGB impaired cell proliferation, but only in the lung adenocarcinoma cell line (H358). Upon hydrogen peroxide treatment, CYGB protected cell viability, migratory potential and anchorage independence by attenuating oxidative injury. In hypoxia, CYGB overexpression decreased cell viability, augmented migration and anchorage independence in a cell-type-specific manner. In conclusion, CYGB revealed TSG properties in normoxia but promoted tumourigenic potential of the cells exposed to stress, suggesting a bimodal function in lung tumourigenesis, depending on cell type and microenvironmental conditions.

Activation of TRPC cationic channels by mercurial compounds confers the cytotoxicity of mercury exposure.

Mercury is an established worldwide environmental pollutant with well-known toxicity affecting neurodevelopment in humans, but the molecular basis of cytotoxicity and the detoxification procedure are still unclear. Here we examined the involvement of the canonical transient receptor potential (TRPC) channel in the mercury-induced cytotoxicity and the potential detoxification strategy. Whole-cell and excised patches, Ca(2+) imaging, and site-directed mutagenesis were used to determine the mechanism of action of mercurial compounds on TRPC channels overexpressed in HEK293 cells, and cytotoxicity and preventive effect were investigated in cell culture models using small interfering RNA and pharmacological blockers. Mercury potently activates TRPC4 and TRPC5 channels. The extracellular cysteine residues (C(553) and C(558)) near the channel pore region of TRPC5 are the molecular targets for channel activation by mercury. The sensitivity of mercury to TRPC5 is presumed to be specific because other divalent heavy metal pollutants, such as Cd(2+), Ni(2+), and Zn(2+), had no stimulating effect, and TRPC3, TRPC6, TRPV1, and TRPM2 were resistant to mercurial compounds. The channel activity of TRPC5, as well as TRPC4, induced by mercury, was prevented by 2-aminoethoxydiphenyl borate and modified by a reducing environment. The inhibition of TRPC5 channels by specific TRPC5 pore-blocking antibody or by SKF-96365 alleviated the cytotoxicity, whereas the mercury chelator, meso-2,3-dimercaptosuccinic acid, showed nonselective prevention of cell survival. Silencing of the TRPC5 gene reduced the mercury-induced neuronal damage. These results indicate that mercurial compounds are activators for TRPC5 and TRPC4 channels. Blockade of TRPC channels could be a novel strategy for preventing mercury-induced cytotoxicity and neurodevelopment impairment.

Neuroglobin and myoglobin in non-small cell lung cancer: expression, regulation and prognosis.

Globins are respiratory proteins involved in oxygen metabolism, which is a critical factor in tumor growth and progression. The status of neuroglobin and myoglobin is largely unknown in human malignancies, including lung cancer. The aim of this study was to explore mRNA expression profiles, potential regulatory mechanisms and clinicopathological associations of neuroglobin and myoglobin in non-small cell lung cancer (NSCLC). We screened 208 surgically resected NSCLC specimens and a panel of lung normal and cancer cell lines. The mRNA expression of neuroglobin, myoglobin and hypoxia markers (HIF1α and VEGFa) was measured with qRTPCR, while neuroglobin promoter methylation was assessed with Pyrosequencing. Neuroglobin and myoglobin were upregulated in the tumor samples compared to normal tissue (p=1.3×10(-22) and p=1.9×10(-9), respectively). Neuroglobin was more frequently overexpressed in squamous cell carcinomas (SqCCL) than adenocarcinomas. Overexpression of myoglobin was more profound in adenocarcinomas, which correlated with poor survival (p=0.013). Neuroglobin promoter was hypermethylated in 30.8% of NSCLC cases, which correlated with neuroglobin mRNA downregulation. The epigenetic regulation of neuroglobin was confirmed by treating lung cell lines with 5'azadeoxycytidine and/or trichostatin A. Expression of both genes correlated with the expression of HIF1α (neuroglobin: p=3.8×10(-5), myoglobin: p=1.1×10(-11)). Myoglobin expression was also associated to that of VEGFa (p=2.1×10(-7)). Hypoxia-dependent upregulation of both globins was validated in vitro. In summary, neuroglobin and myoglobin overexpression in NSCLC is associated with histological subtype, hypoxia and, in case of neuroglobin - epigenetic regulation. Myoglobin expression may have potential significance in the prognostication of lung adenocarcinomas.