Alzheimer disease (AD) is the most common dementia disorder in the elderly. There is substantial evidence that the disease arises from the accumulation of amyloid-beta (Aβ) peptide in the brain, which may form soluble oligomeric species and insoluble Aβ fibrils. The Aβ oligomers have been found to be the most toxic forms of Aβ peptide in neuronal cultures and animal AD models (Lambert et al. 1998; Deshpande et al. 2006; Cizas et al. 2010). The levels of soluble Aβ were found to be increased in brains from AD patients and correlated highly with disease severity (Mclean et al. 1999). Accordingly, Aβ has been suggested as a potential therapeutic target in AD. A number of in vitro and in vivo studies have indicated the beneficial effects of immunotherapy for AD including immunization with Aβ or administration of Aβ antibodies. For example, Aβ antibodies were shown to be effective in reducing the toxicity of Aβ oligomers in cell cultures (Kayed et al. 2003), to prevent Aβ plaque formation and synaptic impairment in transgenic AD animal models (Schenk et al. 1999; Morgan et al. 2001; Hillen et al. 2010) and to slow cognitive decline in human AD patients (Hock et al. 2003).
Several mechanisms have been proposed to account for the protective effects of Aβ antibodies. It has been suggested that anti-Aβ may act in the periphery to bind plasma Aβ leading to an efflux of Aβ from the brain (DeMattos et al. 2001) or if the antibodies cross the blood–brain barrier (BBB), they may trigger Fc-receptor mediated phagocytosis of Aβ (Bard et al. 2000), or they may induce disaggregation of Aβ fibrils (Solomon et al. 1996).
Despite possible beneficial effects, many of the clinical trials of Aβ vaccines were terminated because of the development of meningoencephalitis, an inflammatory reactions in brain, in some AD patients (Nicoll et al. 2003; Orgogozo et al. 2003). In addition, Aβ antibody-induced encephalitis and vasogenic edema have been found to develop in transgenic animal models of AD (Furlan et al. 2003; Minami et al. 2010). The precise mechanism(s) by which anti-Aβ antibodies induce neurotoxicity and/or neuroinflammation remain unclear.
The aim of this study was to elucidate whether complexes of specific antibodies with Aβ aggregates may prevent or cause neurotoxicity in mixed neuronal-glial cultures and whether microglia are involved in this process.
We found that antibodies when in complexes with Aβ or other oligomeric/multimeric antigens exert strong toxic effects on neuronal cells in vitro which are dependent on microglial activation. Our study not only provides new insight into the mechanism of possible Aβ neurotoxicity in the presence of Aβ-specific antibodies in AD brain but may also be important clinically in the development of better and safer vaccines for AD treatment.
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- Materials and methods
- Supporting Information
In this study we show that antigen–antibody complexes formed by Aβ1-42 and specific monoclonal antibodies of IgG isotype can cause death and loss of neurons in primary neuronal-glial cultures by a mechanism that involves microglial activation by the Fc region of antibody–antigen complexes. This conclusion is supported by our findings that the neurotoxic effects of immune complexes were abolished by the removal of microglia from cultures or by removal of the Fc region of #11E12 monoclonal antibody, suggesting that activation of microglial FcγR was required for microglial inflammation and neuronal death. These effects were seen not only with the antibody raised against Aβ1-42 but also with other antibodies raised against recombinant oligomeric antigens – VLPs and NLPs – when they were bound to their target antigens. The neurotoxicity of the antibodies was lost when the same antibodies were applied in complexes with short peptides that do not form oligomeric structures. Therefore, our study demonstrates for the first time that the multimeric structure of antigen–antibody complexes is essential to causing neurotoxicity in mixed neuronal-glial cultures.
Understanding this mechanism may have important implications for developing new strategies for prevention and treatment of AD. The antibody-based immunotherapy to treat AD either via the immunization with Aβ or by the use of Aβ antibodies have shown controversial results in many pre-clinical and clinical studies, however, why the treatment complications such as meningoencephalitis (Nicoll et al. 2003; Orgogozo et al. 2003; Ferrer et al. 2004) does occur in some immunization cases is unclear. The immune response mechanism based on T-cell activation has been suggested which may initiate toxic events (Orgogozo et al. 2003). Passive immunization does not involve T-cell activation, however, significant microglia activation and Fc receptor dependent-phagocytosis was observed in AD patients (Nicoll et al. 2003; Ferrer et al. 2004) and in AD transgenic mice (Bard et al. 2000; Wilcock et al. 2004). Furthermore, auto-antibodies to Aβ were significantly elevated in AD patients and in serum of APP transgenic mice, which have been shown to enhance the neurotoxicity of Aβ (Nath et al. 2003).
Our data are in agreement with these findings and suggest that the neurotoxic effect may result from the interaction of oligomeric complexes of Aβ with antibodies causing Fc receptor mediated activation of microglia. The involvement of microglia in antibody-mediated removal of Aβ aggregates in primary cultures was recently documented for anti-Aβ antibodies (Mohajeri et al. 2006) and human intravenous immunoglobulin IVIG, which contained naturally occurring auto-antibodies, including antibodies against Aβ peptides (Magga et al. 2010). It was shown that peripheral administration of IVIG (Magga et al. 2010) or anti-Aβ antibodies (Koenigsknecht-Talboo and Landreth 2005) caused their penetration into mouse brain tissue and binding to Aβ deposits together with co-localization of microglia. This suggests that peripheral antibodies indeed may cross BBB and interact with the Aβ aggregates and may activate microglia cells. Recently, it has been shown that immune complexes formed in mice brain parenchyma induced a neuroinflammatory response and behavioral changes through recruitment and activation of microglia via FcγR (Teeling et al. 2012). Activated microglia, via production of reactive oxygen species and other inflammatory mediators, may be sufficient to induce neuronal death (Brown and Neher 2010) and neuronal loss (Neniskyte et al. 2011).
We have previously determined that the epitope of #11E12 antibody was localized at the N-terminus of Aβ1-42, between amino acid residues 1 and 6, and that #11E12 antibody reacted with the peptide representing the N terminal sequence (aa 1–13) (Dalgediene et al. 2013). However, the complexes of #11E12 antibody plus Aβ1-6 or #11E12 antibody plus Aβ1-13 were not toxic to cultured neurons. This indicates that neurotoxicity of the antibody–antigen complex is depended on the structure of antigen. Multimeric antigens may bind multiple antibodies, which may bring together multiple FcγR and thereby activate them to induce inflammation. Whereas monomeric antigens may bind single antibodies recruiting single FcγR resulting in minimal inflammation. Therefore, development of short Aβ immunogens that target monomeric Aβ may be a promising approach for AD treatment.
We also showed that the cytotoxic effects of antibody–antigen complexes disappeared when microglia cells were removed from the culture or when the Fc part of the antibody was cleaved off, indicating the involvement of microglia FcγR. These data are consistent with results obtained with deglycosylated anti-Aβ, which were shown to reduce microglia phagocytosis and cytokine production because of reduced affinity to FcγR in primary cultured microglia (Takata et al. 2007). In addition, deglycosylated anti-Aβ (Carty et al. 2006) or F(ab')2 fragments (Tamura et al. 2005) were able to suppress the microhemorrhages in AD mice. In accordance, our data showed that minocycline, which is known to inhibit microglial activation in animal models of neurodegenerative diseases (Fan et al. 2007), was neuroprotective against anti-Aβ + Αβ induced release of TNF-α and neuronal death.
Rodent microglia cells have four types of FcγR (Ravetch and Kinet 1991; Nimmerjahn et al. 2005). The IgG subclass specificities of mouse and rat phagocytic cells FcγR are likely to be more similar to each other (Song et al. 1990). FcγRII lack phagocytic activity (Takai et al. 1994), however, FcγRI, FcγRIII, and FcγRIV exhibit affinity to mouse IgG (Fossati-Jimack et al. 2000; Nimmerjahn et al. 2005). All three subtypes of IgG were able to reduce Aβ deposits in mouse microglia cultures (Bard et al. 2003). Moreover, the deleterious effects of anti-Aβ in AD mice also have been obtained with different IgG subtypes: microhemorrhage with IgG2a (Schroeter et al. 2008), encephalomyelitis with IgG1, IgG2a (Furlan et al. 2003), and IgG2b (Carty et al. 2006), microglia activation with IgG1 (Wilcock et al. 2004). We used primary rat and mouse cultures and found cytotoxic effects of different mouse IgG subtype complexes, IgG2b (anti-Aβ, clone #11E12, anti-MTP-N, clone #4A2 and anti-MeN, #10F7), IgG2a (anti-VP1,clone #6D11), and IgG1 (anti-Aβ42, clone #G2-13) indicating that neurotoxic effect was not animal species dependent as mouse antibody complexes were similarly toxic to rat as well as to mouse neurons. However, the capacity of FcγR to bind various IgG subtypes may be different, the high-affinity FcγRI is capable to bind preferentially IgG2a (Sears et al. 1990), the low-affinity FcγRIII binds IgG1, IgG2a, and IgG2b (Weinshank et al. 1988), and a recently identified high-affinity FcγRIV binds IgG2a and IgG2b (Nimmerjahn et al. 2005; Bruhns 2012). These divergent activities might have contribution to antibody-mediated cytotoxicity (Nimmerjahn and Ravetch 2006). Indeed, our study suggests that rat microlia FcγR can bind oligomeric antigen–antibody of different mouse IgG isotypes and initiate the sequence of toxic reactions at different level. In contrast, the complexes of polyclonal rabbit anti-Aβ42 were non-toxic to cultured neurons suggesting that rat microglia do not recognize rabbit antibodies possibly because of a low affinity for rat FcγR.
We used two different monoclonal Aβ1-42 antibodies, the clone #11E12, that targeted N-terminal epitope and clone #G2-13, that targeted the C-terminus, and both in complexes with Aβ1-42 oligomers induced death of neurons in cultures indicating that the epitope position does not play any role in oligomeric antigen–antibody complex-induced cytotoxicity. Our results are in agreement with those obtained in passive immunization studies where microhemorrhage appearance and microglia activation followed treatment with N-terminal Aβ antibodies (Racke et al. 2005) as well as after C-terminal Aβ antibodies (Carty et al. 2006).
We did not find any significant differences in induction of neuronal death when either Aβ1-42 oligomers, fibrils, and monomers bound to the specific monoclonal antibody #11E12 were used most likely because of rapid oligomerization/fibrilization of Aβ1-42 monomers in aqueous solutions. However, our data revealed the difference in toxicity caused by complexes of #11E12 antibody with monomeric and oligomeric forms of Aβ1-40. The aggregation of Aβ1-40 monomers has been shown to be slow in aqueous media (hours to days) in contrast to rapid aggregation (minutes) of Aβ1-42 (Snyder et al. 1994), therefore, the Aβ1-40 monomeric preparation were likely to be in the monomeric state during experiments and therefore did not cause neurotoxicity when complexed with antibodies.
Our data also demonstrate that various oligomeric proteins when complexed with specific antibodies such as the antibodies raised against recombinant VLPs and NLPs can induce microglial Fc-receptor dependent death of cultured neurons. In contrast, complexes of short linear epitope MeN440-452 bound to anti-MeN were non-toxic to neurons. This confirms the finding that antigen oligomeric conformation of immune complexes is crucial in causing cell death in vitro. Such mechanism may be involved in neuroinflammatory responses caused by various viral infections particularly in the cases of disruption of BBB leading to penetration of viral oligomeric proteins and virus-specific antibodies into brain. Human polyomaviruses include the JC virus that infects the brain causing progressive multifocal leukoencephalopathy (PML) accompanied by brain inflammation and neuronal loss. Measles virus can also infect the brain causing acute encephalitis or more rarely subacute sclerosing panencephalitis, driven by brain inflammation and neuronal loss (Hayashi et al. 2002). If endogenous antibodies to oligomeric viral proteins enter the brain, they might cause neuroinflammation and neurotoxicity by the mechanism described here, and if so pathology might be limited by inhibiting microglial activation e.g. with minocycline.
The data presented here demonstrate that oligomeric/multimeric antigens complexed with their specific antibodies can exert cytotoxic effects on primary neurons by Fc-dependent microglia activation. Thus, if these antibodies cross the BBB and bind their oligomeric/multimeric antigens in the brain, they may induce microglia activation and neurotoxicity by this mechanism. Elucidation of the detailed mechanism of antibody–antigen complex-mediated neuroinflammation may help limit the neurotoxicity and potentially increase the effectiveness of immunotherapy for AD.