- Email: [email protected]
A new strategy for protection of neurons from complement-mediated degeneration triggered by β-amyloid plaques
4116
Abstracts / Molecular Immunology 45 (2008) 4095–4182
lion years ago. Cnidarians lack the third germ layer ‘mesoderm’ as well as the body cavity, the main site for the activity of the complement system in all bilateria analyzed so far. Here we performed the comprehensive cloning and expression analyses of the complement genes in a cnidarian sea anemone, Nematostella vectensis. The draft genome database of N. vectensis was BLAST-searched using the sequences of the human complement components having unique domain structure, C3, factor B (Bf), MASP, C6 and factor I (If). As a result, two C3, two Bf and one MASP genes were identified, whereas no C6 or If gene was identified. The entire protein-coding sequences of the identified five genes were elucidated by the RT-PCR and 5(-, 3(-RACE-PCR, and their deduced protein domain structures were completely identical with that of their mammalian counterparts, including signal peptide. Moreover, the cnidarian complement components retained almost all the functionally critical amino acid residues, suggesting the conservation of the basic biochemical functions. In situ hybridization analysis indicated that all five genes are expressed at the tip of tentacles, pharynx and mesentery in an endoderm-specific manner. These results indicate that the complement system composed of at least three components, C3, Bf and MASP, was established prior to the divergence of cnidaria and bilateria. The cnidarian complement components are produced in endoderm, and are implicated in the defense mechanism against pathogens taken up with foods or water into coelenteron, a digestive cavity of cnidaria with circulatory function. doi:10.1016/j.molimm.2008.08.061 Complement in health and disease O61 Measurement of factor H and the Y402H polymorphism in Alzheimer’s disease Svetlana Hakobyan, Claire Abraham, B. Paul Morgan
L.
Harris, Julie
Williams, Richard
School of Medicine, Cardiff University, Cardiff, UK Evidence is accumulating that chronic inflammation occurs in the Alzheimer’s disease (AD) brain and contributes significantly to neuronal loss and resultant cognitive decline. A consistent feature of chronic inflammatory disease is activation of the alternative pathway (AP) of C, an antibody-independent event that exacerbates local injury and inflammation. Healthy tissue is protected by regulatory proteins present on cells and in fluids, but in disease regulation fails. C activation has been observed in AD brain although its contribution to the disease process is poorly defined; importantly, factor H (fH), the major regulator of the AP, is also found in AD plaques suggesting that regulation occurs at this site. Mutations and polymorphisms in fH have recently been associated with chronic inflammatory diseases of the kidney and eye, suggesting that subtle changes in this protein might “dysregulate” the AP and predispose to disease at particular sites. Recently, a proteomic screen showed that plasma levels of fH, assessed in a semi-quantitative manner, were elevated in AD and correlated with disease activity. We have described quantitative assays for total fH and the Y402H variants and here apply them to plasma samples from AD patients. Compared to non-demented matched controls, AD patients had markedly elevated total fH levels (mean 394 mg/l vs. 269 mg/l for controls). Intriguingly, the H402 allele frequency was higher in AD patients compared with controls, and in heterozygote AD patients but not controls the plasma levels of the H402 variant were significantly reduced compared to the Y402 variant, suggesting differential expression or consumption.
These data confirm and extend the observation that plasma levels of fH are elevated in AD. We suggest that alterations in levels and/or expression of fH and its variants may be implicated in pathology in AD and may be biomarkers of disease, predictors of disease activity and course and guides to therapy. doi:10.1016/j.molimm.2008.08.062 O62 The two AMD-associated variants of the factor H protein (V62I and Y402H) affect protein function and complement control Nadine Lauer a , Andrea Hartmann a , Julia Böhme a , Steffi Hälbich a , Frank Sühnel b , Ursula Schlötzer-Schrehardt c , Christine Skerka a , Peter F. Zipfel a a
Hans-Knöll-Institue, Jena, Germany Leibniz Institute for Age Research, Jena, Germany c University Hospital of Erlangen, Erlangen, Germany b
Age-related macular degeneration (AMD) is the most common cause of blindness in the elderly population of the Western World and affects about 50 million people worldwide. The complement system is involved in the pathogenesis of this retinal disease and several polymorphisms in the factor H gene are associated with AMD. The most prominent are the V62I and Y402H variants of the factor H protein, which increase the risk for AMD about 50%. Here we analyzed the role of the two common AMD-associated variants of Factor H and FHL-1: V62I and Y402H. Factor H with these substitutions was purified from plasma of genotyped patients and FHL-1 variants were generated by in vitro mutagenesis and recombinantly expressed. With this approach we compared four protein variants: V62-Y402; V62-H402; I62-Y402; I62-H402 and show, that the binding site within SCR 7 interacts specifically with the monomeric form of C-reactive protein (mCRP). The H402 variants of both, factor H and FHL-1 showed reduced binding to mCRP as demonstrated by ELISA and surface plasmon resonance experiments. The lower affinity of both complement regulators impairs mCRP function and results in reduced recruitment of factor H and FHL-1 to the surface of necrotic cells. The V62I substitution within factor H and FHL-1 affects C3b binding which results in a decelerated cofactor activity in fluid phase. In summary the two common variants V62I and Y402H of the two complement regulators factor H and FHL-1 lead to a functional impairment of complement control in fluid phase and on the surface of intact, as well as necrotic retinal pigment epithelial cells. A combination of both risk residues I62 and H402 further reduces complement regulation. This effect can explain how inadequate complement control on retinal surfaces can enhance local inflammation and thus may lead to AMD progression. doi:10.1016/j.molimm.2008.08.063 O63 A new strategy for protection of neurons from complementmediated degeneration triggered by -amyloid plaques Martin Kolev, Teeo Tediose, B. Paul Morgan, Rossen Donev Cardiff University, Cardiff, UK Large numbers of neuritic plaques, predominantly composed of a fibrillar insoluble form of the -amyloid peptide (Abeta), are found in the hippocampus and neocortex of Alzheimer’s disease (AD) patients in association with damaged neuronal processes. Complement (C) proteins are integral components of amyloid
Abstracts / Molecular Immunology 45 (2008) 4095–4182
4117
plaques and cerebral vascular amyloid in AD brains. They are found at the earliest stages of amyloid deposition and their activation coincides with the clinical expression of Alzheimer’s dementia. Amyloid plaques activate C via binding C1q and C3b resulting in Cmediated neurodegeneration. The aim of this work was to develop a strategy to increase the resistance of neurons to C damage by modulating expression of membrane C regulatory proteins. It has been shown previously in different model systems of neurons that cerebrolysin (CBL), an anti-apoptotic peptide-based drug that exhibits neurotrophic and neuroprotective activity, reduces accumulation of the amyloid plaques. Here, for the first time, we demonstrate this effect in neurons differentiated from human neural progenitor cells and further show that CBL upregulated expression of the membrane-bound C regulators CD59 and CD55, but CD46 remained unchanged. CD59 and CD55 expression protected neurons from C lysis triggered by amyloid plaques. We have further exploited our recent finding about regulation of CD59 expression by the neural-restrictive silencer factor (REST) and designed a REST-derived peptide (REST5), comprising the nuclear localisation domain of the wild type transcription factor, that increased expression of CD59 in neurons by 5-fold. Treatment of neurons with REST5 alone protected them from C-mediated lysis, while in combination with CBL we observed a synergistic effect resulting in a higher protection of neurons from C killing. This study brings a new insight into pharmacological therapies that may serve to inhibit Abeta-mediated C activation.
the development of pre-eclampsia. Analysis included multivariable logistic regression. The incidence of pre-eclampsia was 4.6%. The adjusted odds ratio of Bb (top decile), PlGF (lower decile) and sFlt-1 (top decile) for pre-eclampsia were 5.5 (95% CI 2–15.5, P = 0.001), 5.0 (CI 1.8–14, P = 0.002) and 3.6 (CI 1.2–11, P = 0.03), respectively. Significantly higher levels of Bb were found among women with a BMI ≥30 as compared with women with lower BMI levels (P < 0.001). In contrast, levels of sFlt-1 levels were significantly higher among women in the lower categories of BMI (P < 0.001 for trend). Complement Bb in early pregnancy remains an independent risk factor for pre-eclampsia even after adjustment for potential confounders including angiogenesis-related factors. We are currently evaluating whether complement fragments derived from C3 and/or C5 activation are related to early changes in angiogenic factor levels.
doi:10.1016/j.molimm.2008.08.064
We have recently shown that endothelial cells (ECs) isolated from human decidua synthesize C1q and express surface-bound C1q under physiological conditions [Bulla et al., 2008. Mol. Immunol.]. Since decidua is a typical site of vascular remodelling and active angiogenic process, we sought to ascertain whether C1q may play a role in promoting angiogenesis using HUVEC in a number of experimental approaches. First, we tested the ability of C1q to induce leakage of FITC-BSA through a monolayer of ECs, as a measure of increased permeability, which is currently considered an initial event of angiogenesis. C1q induced a permeabilizing effect comparable to that of bradykinin, used as a positive control. Next, we found that C1q was able to induce EC proliferation and to act as a chemotactic factor recruiting ECs in a transwell model system. C1q also promoted the motility of ECs in the wound healing assay as evaluated by the ability of these cells to cover the scratched area of an EC monolayer. To further assess whether C1q may stimulate the formation of new vessels, we used an in vitro assay of tube formation, in which ECs are grown in a tridimensional matrix of matrigel to allow the assembly of capillary network. Under these conditions, C1q proved to be as effective as VEGF in inducing tube formation. The pro-angiogenic effect of C1q was also tested in the rat aortic ring assay evaluating the sprouting and branching of new vessels. C1q was found to induce a comparable number of new vessels as VEGF, but with more branches. Taken together, these data disclose an unexpected pro-angiogenic effect of C1q.
O64 Complement activation fragment Bb, angiogenic factors and pre-eclampsia Anne Lynch a , James Murphy b , Richard Levine c , Ronald Gibbs a , Patricia Giclas d , Tim Byers e , Mark Parish a , Jane Salmon f , V. Michael Holers a a
University of Colorado, Denver, Aurora, USA National Jewish Medical and Research Center, Denver, USA c National Institute of Child Health and Development, NIH, Bethesda, USA d Pediatrics Department, National Jewish Medical and Research Center, Denver, USA e Department of Epidemiology, Colorado School of Public Health, Aurora, USA f Hospital for Special Surgery, Weill Medical College, Cornell University, New York, USA b
Immune-mediated events in the placenta and altered expression of placental angiogenic factors contribute to the pathogenesis of pre-eclampsia, a syndrome characterized by an increase in maternal blood pressure in the second half of pregnancy accompanied by proteinuria. We have demonstrated that elevated levels of the alternative complement pathway activation fragment Bb in early pregnancy are associated with subsequent development of pre-eclampsia. Other research from murine models of pregnancy loss has shown that complement activation is a trigger for the production of antiangiogenic factors. Our objective was to determine whether Bb in early pregnancy is an independent risk factor for pre-eclampsia after adjustment for angiogenesis-related factors, including placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and maternal risk factors. We conducted a prospective study of 647 women in Denver, Colorado. A single plasma sample was obtained from each woman between 10 and 20 weeks gestation. The cohort was followed for
doi:10.1016/j.molimm.2008.08.065 O65 C1q: A novel angiogenic factor? Fleur Bossi a , Lucia Rizzi a , Roberta Bulla a , Carine Munaut b , Stefania Berton c , Gustavo Baldassarre c , Francis Frankenne b , Francesco Tedesco a a
University of Trieste, Trieste, Italy Laboratory of Tumor Biology and Development, Liège, Belgium c CRO, Aviano, Italy b
doi:10.1016/j.molimm.2008.08.066
Abstracts / Molecular Immunology 45 (2008) 4095–4182
lion years ago. Cnidarians lack the third germ layer ‘mesoderm’ as well as the body cavity, the main site for the activity of the complement system in all bilateria analyzed so far. Here we performed the comprehensive cloning and expression analyses of the complement genes in a cnidarian sea anemone, Nematostella vectensis. The draft genome database of N. vectensis was BLAST-searched using the sequences of the human complement components having unique domain structure, C3, factor B (Bf), MASP, C6 and factor I (If). As a result, two C3, two Bf and one MASP genes were identified, whereas no C6 or If gene was identified. The entire protein-coding sequences of the identified five genes were elucidated by the RT-PCR and 5(-, 3(-RACE-PCR, and their deduced protein domain structures were completely identical with that of their mammalian counterparts, including signal peptide. Moreover, the cnidarian complement components retained almost all the functionally critical amino acid residues, suggesting the conservation of the basic biochemical functions. In situ hybridization analysis indicated that all five genes are expressed at the tip of tentacles, pharynx and mesentery in an endoderm-specific manner. These results indicate that the complement system composed of at least three components, C3, Bf and MASP, was established prior to the divergence of cnidaria and bilateria. The cnidarian complement components are produced in endoderm, and are implicated in the defense mechanism against pathogens taken up with foods or water into coelenteron, a digestive cavity of cnidaria with circulatory function. doi:10.1016/j.molimm.2008.08.061 Complement in health and disease O61 Measurement of factor H and the Y402H polymorphism in Alzheimer’s disease Svetlana Hakobyan, Claire Abraham, B. Paul Morgan
L.
Harris, Julie
Williams, Richard
School of Medicine, Cardiff University, Cardiff, UK Evidence is accumulating that chronic inflammation occurs in the Alzheimer’s disease (AD) brain and contributes significantly to neuronal loss and resultant cognitive decline. A consistent feature of chronic inflammatory disease is activation of the alternative pathway (AP) of C, an antibody-independent event that exacerbates local injury and inflammation. Healthy tissue is protected by regulatory proteins present on cells and in fluids, but in disease regulation fails. C activation has been observed in AD brain although its contribution to the disease process is poorly defined; importantly, factor H (fH), the major regulator of the AP, is also found in AD plaques suggesting that regulation occurs at this site. Mutations and polymorphisms in fH have recently been associated with chronic inflammatory diseases of the kidney and eye, suggesting that subtle changes in this protein might “dysregulate” the AP and predispose to disease at particular sites. Recently, a proteomic screen showed that plasma levels of fH, assessed in a semi-quantitative manner, were elevated in AD and correlated with disease activity. We have described quantitative assays for total fH and the Y402H variants and here apply them to plasma samples from AD patients. Compared to non-demented matched controls, AD patients had markedly elevated total fH levels (mean 394 mg/l vs. 269 mg/l for controls). Intriguingly, the H402 allele frequency was higher in AD patients compared with controls, and in heterozygote AD patients but not controls the plasma levels of the H402 variant were significantly reduced compared to the Y402 variant, suggesting differential expression or consumption.
These data confirm and extend the observation that plasma levels of fH are elevated in AD. We suggest that alterations in levels and/or expression of fH and its variants may be implicated in pathology in AD and may be biomarkers of disease, predictors of disease activity and course and guides to therapy. doi:10.1016/j.molimm.2008.08.062 O62 The two AMD-associated variants of the factor H protein (V62I and Y402H) affect protein function and complement control Nadine Lauer a , Andrea Hartmann a , Julia Böhme a , Steffi Hälbich a , Frank Sühnel b , Ursula Schlötzer-Schrehardt c , Christine Skerka a , Peter F. Zipfel a a
Hans-Knöll-Institue, Jena, Germany Leibniz Institute for Age Research, Jena, Germany c University Hospital of Erlangen, Erlangen, Germany b
Age-related macular degeneration (AMD) is the most common cause of blindness in the elderly population of the Western World and affects about 50 million people worldwide. The complement system is involved in the pathogenesis of this retinal disease and several polymorphisms in the factor H gene are associated with AMD. The most prominent are the V62I and Y402H variants of the factor H protein, which increase the risk for AMD about 50%. Here we analyzed the role of the two common AMD-associated variants of Factor H and FHL-1: V62I and Y402H. Factor H with these substitutions was purified from plasma of genotyped patients and FHL-1 variants were generated by in vitro mutagenesis and recombinantly expressed. With this approach we compared four protein variants: V62-Y402; V62-H402; I62-Y402; I62-H402 and show, that the binding site within SCR 7 interacts specifically with the monomeric form of C-reactive protein (mCRP). The H402 variants of both, factor H and FHL-1 showed reduced binding to mCRP as demonstrated by ELISA and surface plasmon resonance experiments. The lower affinity of both complement regulators impairs mCRP function and results in reduced recruitment of factor H and FHL-1 to the surface of necrotic cells. The V62I substitution within factor H and FHL-1 affects C3b binding which results in a decelerated cofactor activity in fluid phase. In summary the two common variants V62I and Y402H of the two complement regulators factor H and FHL-1 lead to a functional impairment of complement control in fluid phase and on the surface of intact, as well as necrotic retinal pigment epithelial cells. A combination of both risk residues I62 and H402 further reduces complement regulation. This effect can explain how inadequate complement control on retinal surfaces can enhance local inflammation and thus may lead to AMD progression. doi:10.1016/j.molimm.2008.08.063 O63 A new strategy for protection of neurons from complementmediated degeneration triggered by -amyloid plaques Martin Kolev, Teeo Tediose, B. Paul Morgan, Rossen Donev Cardiff University, Cardiff, UK Large numbers of neuritic plaques, predominantly composed of a fibrillar insoluble form of the -amyloid peptide (Abeta), are found in the hippocampus and neocortex of Alzheimer’s disease (AD) patients in association with damaged neuronal processes. Complement (C) proteins are integral components of amyloid
Abstracts / Molecular Immunology 45 (2008) 4095–4182
4117
plaques and cerebral vascular amyloid in AD brains. They are found at the earliest stages of amyloid deposition and their activation coincides with the clinical expression of Alzheimer’s dementia. Amyloid plaques activate C via binding C1q and C3b resulting in Cmediated neurodegeneration. The aim of this work was to develop a strategy to increase the resistance of neurons to C damage by modulating expression of membrane C regulatory proteins. It has been shown previously in different model systems of neurons that cerebrolysin (CBL), an anti-apoptotic peptide-based drug that exhibits neurotrophic and neuroprotective activity, reduces accumulation of the amyloid plaques. Here, for the first time, we demonstrate this effect in neurons differentiated from human neural progenitor cells and further show that CBL upregulated expression of the membrane-bound C regulators CD59 and CD55, but CD46 remained unchanged. CD59 and CD55 expression protected neurons from C lysis triggered by amyloid plaques. We have further exploited our recent finding about regulation of CD59 expression by the neural-restrictive silencer factor (REST) and designed a REST-derived peptide (REST5), comprising the nuclear localisation domain of the wild type transcription factor, that increased expression of CD59 in neurons by 5-fold. Treatment of neurons with REST5 alone protected them from C-mediated lysis, while in combination with CBL we observed a synergistic effect resulting in a higher protection of neurons from C killing. This study brings a new insight into pharmacological therapies that may serve to inhibit Abeta-mediated C activation.
the development of pre-eclampsia. Analysis included multivariable logistic regression. The incidence of pre-eclampsia was 4.6%. The adjusted odds ratio of Bb (top decile), PlGF (lower decile) and sFlt-1 (top decile) for pre-eclampsia were 5.5 (95% CI 2–15.5, P = 0.001), 5.0 (CI 1.8–14, P = 0.002) and 3.6 (CI 1.2–11, P = 0.03), respectively. Significantly higher levels of Bb were found among women with a BMI ≥30 as compared with women with lower BMI levels (P < 0.001). In contrast, levels of sFlt-1 levels were significantly higher among women in the lower categories of BMI (P < 0.001 for trend). Complement Bb in early pregnancy remains an independent risk factor for pre-eclampsia even after adjustment for potential confounders including angiogenesis-related factors. We are currently evaluating whether complement fragments derived from C3 and/or C5 activation are related to early changes in angiogenic factor levels.
doi:10.1016/j.molimm.2008.08.064
We have recently shown that endothelial cells (ECs) isolated from human decidua synthesize C1q and express surface-bound C1q under physiological conditions [Bulla et al., 2008. Mol. Immunol.]. Since decidua is a typical site of vascular remodelling and active angiogenic process, we sought to ascertain whether C1q may play a role in promoting angiogenesis using HUVEC in a number of experimental approaches. First, we tested the ability of C1q to induce leakage of FITC-BSA through a monolayer of ECs, as a measure of increased permeability, which is currently considered an initial event of angiogenesis. C1q induced a permeabilizing effect comparable to that of bradykinin, used as a positive control. Next, we found that C1q was able to induce EC proliferation and to act as a chemotactic factor recruiting ECs in a transwell model system. C1q also promoted the motility of ECs in the wound healing assay as evaluated by the ability of these cells to cover the scratched area of an EC monolayer. To further assess whether C1q may stimulate the formation of new vessels, we used an in vitro assay of tube formation, in which ECs are grown in a tridimensional matrix of matrigel to allow the assembly of capillary network. Under these conditions, C1q proved to be as effective as VEGF in inducing tube formation. The pro-angiogenic effect of C1q was also tested in the rat aortic ring assay evaluating the sprouting and branching of new vessels. C1q was found to induce a comparable number of new vessels as VEGF, but with more branches. Taken together, these data disclose an unexpected pro-angiogenic effect of C1q.
O64 Complement activation fragment Bb, angiogenic factors and pre-eclampsia Anne Lynch a , James Murphy b , Richard Levine c , Ronald Gibbs a , Patricia Giclas d , Tim Byers e , Mark Parish a , Jane Salmon f , V. Michael Holers a a
University of Colorado, Denver, Aurora, USA National Jewish Medical and Research Center, Denver, USA c National Institute of Child Health and Development, NIH, Bethesda, USA d Pediatrics Department, National Jewish Medical and Research Center, Denver, USA e Department of Epidemiology, Colorado School of Public Health, Aurora, USA f Hospital for Special Surgery, Weill Medical College, Cornell University, New York, USA b
Immune-mediated events in the placenta and altered expression of placental angiogenic factors contribute to the pathogenesis of pre-eclampsia, a syndrome characterized by an increase in maternal blood pressure in the second half of pregnancy accompanied by proteinuria. We have demonstrated that elevated levels of the alternative complement pathway activation fragment Bb in early pregnancy are associated with subsequent development of pre-eclampsia. Other research from murine models of pregnancy loss has shown that complement activation is a trigger for the production of antiangiogenic factors. Our objective was to determine whether Bb in early pregnancy is an independent risk factor for pre-eclampsia after adjustment for angiogenesis-related factors, including placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and maternal risk factors. We conducted a prospective study of 647 women in Denver, Colorado. A single plasma sample was obtained from each woman between 10 and 20 weeks gestation. The cohort was followed for
doi:10.1016/j.molimm.2008.08.065 O65 C1q: A novel angiogenic factor? Fleur Bossi a , Lucia Rizzi a , Roberta Bulla a , Carine Munaut b , Stefania Berton c , Gustavo Baldassarre c , Francis Frankenne b , Francesco Tedesco a a
University of Trieste, Trieste, Italy Laboratory of Tumor Biology and Development, Liège, Belgium c CRO, Aviano, Italy b
doi:10.1016/j.molimm.2008.08.066