To experimentally address the role of complement C3 in the pathogenesis of AD, we generated C3-deficient APP transgenic mice and analyzed them neuropathologically and biochemically at 8, 12 and 17 months of age, comparing them to age-matched APP transgenic mice. At younger ages (8 and 12 months), no significant differences were observed between APP and APP;C3−/−
mice in plaque load, biochemical levels of Aβ, or in any of the neuronal or glial markers examined. In contrast, at 17 months of age, the APP;C3−/−
mice showed a significant ~two-fold increase in total Aβ and fibrillar amyloid plaque burden that correlated with significantly increased guanidine soluble Aβ42 levels and reduced TBS soluble Aβ42 and Aβ40 in brain homogenates, a non-significant trend for increased plasma Aβ levels, a significant reduction of NeuN positive neurons in the CA3 region of the hippocampus, and a shift of microglia/macrophage towards a more alternative activation/M2 phenotype (according to Mantovani et al., 2004
; Morgan et al., 2005
). Our findings demonstrate that the complete absence of the central complement component, C3, accelerates AD-like plaque pathology with aging once plaque pathogenesis is underway. Although many of the differences between the APP:C3−/−
and APP mice reported here were statistically significant, many comparisons resulted in a non-significant trend due to the relatively small number of mice studied and the high variability observed in APP transgenic mice, in general. However, these trends provide additional support to the overall findings.
The rather late effect of C3-deficiency may be related to an attempt of APP mice to protect the brain by upregulating complement C3 once AD-like pathogenesis is underway. Similar to a previous report by Wyss-Coray and colleagues (2002)
, we observed an increase in C3 protein levels with aging and AD pathogenesis in APP mice. In their study, Wyss-Coray and colleagues demonstrated that C3 mRNA levels increased with age in APP mice, and that C3 protein levels were elevated when TGFβ was overexpressed in APP mice (Wyss-Coray et al., 2002
). Inhibition of complement C3 convertase (to block activation of C3) by overexpression of sCrry in APP mice resulted in increased plaque burden and neurodegeneration, even in the presence of C3 protein (Wyss-Coray, 2002
Consistent with an important role of complement in late stage AD-like pathogenesis is a study (Matsuoka et al., 2001
) showing the colocalization of complement-activating C1q and an upregulation of C1q with increased formation of fibrillar Aβ plaques in a PS1/APP transgenic mouse model. In addition, we previously reported prominent C1q and C3 immunoreactivity in highly compacted Aβ42-positive neuritic plaques associated with microgliosis in the cortex of middle-aged and older individuals with Down syndrome (Stoltzner et al., 2000
). Whether the elevation of complement proteins in AD brain is an attempt to protect the brain or a consequence of neuronal damage is unclear but our results, along with those of Wyss-Coray (2002)
, suggest that complement C3 may play a protective role in the brain.
Amyloid fibrils have been detected in microglia in human AD brain suggesting that microglia are involved in the clearing of Aβ protein deposits (Wegiel and Wisniewski, 1990
). Furthermore, it has also been shown in multiple studies that different forms of aggregated Aβ activate and become bound by complement opsonins such as C3b (Webster et al., 1997
; Bradt et al., 1998
) which facilitates CR3-mediated phagocytosis of Aβ (Ehlers, 2000
). Recent mouse studies report a reduction of the microglial markers F4/80 and I-A/I-E (marker of MHCII alloantigen) in old C1q-deficient Tg2576 (APP) mice (Fonseca et al., 2004
). In addition, F4/80-positive microglia were reduced in APP/sCrry mice (Wyss-Coray et al., 2002
). By Western blot, we, too, found a non-significant trend for reduced F4/80 as well as CD68 levels in our 17 month-old APP;C3−/−
mice. Therefore, it is possible that the increased fibrillar plaque load observed in our C3-deficient APP tg mice may be due to less efficient phagocytosis of Aβ fibrils in the absence of C3 due to lack of C3b or iC3b- mediated opsonization.
Interestingly, although F4/80 and CD68 levels were reduced in our 17 month-old APP;C3−/−
mice compared to age-matched APP mice, we found increases in other microglia/macrophage markers of activation including CD45 (significant) and Iba1 (non-significant trend), correlating with increased Aβ plaque burden and neurodegeneration. C3-deficiency in 17 month-old APP mice resulted in increased IL-4 (p<0.05 in TBS soluble fraction) and IL-10 (non-significant trend) and, reduced iNOS (p<0.05) and TNF (p<0.05 in TBS-T membrane-bound fraction) brain levels. Others have demonstrated that anti-inflammatory cytokines, such as IL-4 and IL-10, increased fibrillar Aβ phagocytosis by murine primary microglia in vitro
(Koenigsknecht-Talboo et al., 2005
). Although anti-inflammatory cytokine levels were elevated in the APP;C3−/−
mice in our study, Aβ deposition was increased as well, indicating that Aβ phagocytosis by microglia was ineffective at removing Aβ deposits. Thus, it is possible that the presence of complement C3 may be necessary for anti-inflammatory cytokines to stimulate microglial phagocytosis of aggregated Aβ.
Taken together, our findings indicate a shift of the microglia/macrophage response towards an alternative M2 activation phenotype (Manotovani et al., 2004; Morgan et al., 2005
) in the absence of complement C3. M2-type microglia/macrophage are often found in association with apoptotic cells to scavenge debris and promote tissue repair which is in agreement with the increased neurodegeneration we observed in the 17 month-old APP;C3−/−
mice. Upregulation of CD45, which is expressed at high levels in infiltrating microglia/macrophage (Ford et al., 1995
), could represent enhanced infiltration of peripheral macrophages due to increased fibrillar Aβ deposits and a higher incidence of dying cells in brain. It should be noted that in the study of Wyss-Coray et al. (2002)
inhibition of C3 convertase by sCrry overexpression may have only affected certain microglial functions such as Aβ phagocytosis, while the complete absence of C3 may have additional affects on microglial function and/or molecules that normally suppress microglial activation along the phagocytic and cytotoxic pathways. Ours is the first study to fully examine the role of complement C3 on the microglia/macrophage phenotype and cytokines in APP mice.
Alternatively, increased Aβ deposition in brain and Aβ levels in plasma in C3-deficient APP tg mice may also be due to reduced peripheral degradation of Aβ. Indeed, a recent study suggested an important role of complement C3 in the peripheral clearance of Aβ by C3b-dependent adherence to complement receptor 1 (CR1) on erythrocytes in blood of humans (Rogers et al., 2006
). A recently described complement receptor, CR1g, was found to be important for C3b-dependent clearance of pathogens from the blood (Helmy et al., 2006
) and thus, may represent an additional pathway for clearance of complement-opsonized Aβ in the periphery. Such a mechanism should be affected in C3-deficient APP mice. Indeed, the absence of C3 resulted in the accumulation of Aβ in the periphery, thereby increasing the amount of peripheral Aβ available for influx into the brain.
In contrast to guanidine-soluble Aβ42 levels which were increased with the fibrillar Aβ plaque load, TBS-soluble and to some extent also TBS-T soluble Aβ levels were reduced in the brains of C3-deficient APP mice. Therefore, it is possible that complement C3 also plays a role in suppressing aggregation of Aβ in which case, the lack of C3 would result in reduced soluble Aβ levels in brain and increased deposition of aggregated Aβ into plaques. However, this seems unlikely as C3 levels were elevated in the complement-sufficient APP tg mice with age as Aβ became more aggregated.
Increased amounts of fibrillar Aβ deposition in 17 month-old APP;C3−/−
mice correlated with a reduction of NeuN positive neurons in hippocampus, suggesting a role for complement C3 in neuronal survival and health. Neuronal loss was highest in CA3 compared to CA1, possibly due to increased hAPP transgene expression and Aβ deposition. Neurons express C3 thus the neurons in C3-deficient APP mice may be more vulnerable to the cytotoxic effects of Aβ and/or microglial activation. However, C3-deficient neurons in 12 month-old APP;C3−/−
mice appeared relatively healthy. MAP2 and synaptophysin immunoreactivity were non-significantly reduced in APP;C3−/−
mice, in agreement with the findings of Wyss-Coray (2002)
in which a similar neuronal phenotype was observed upon inhibition of C3 convertase. By electron microscopy, they reported an accumulation of degenerating neurons in APP/sCrry mice compared to wildtype mice that correlated with the reduction of NeuN-positive neurons. Our findings agree with other studies that have proposed a neuroprotective effect of complement proteins and complement activation products such as C3a and C5a (van Beek et al., 2003
A recent study attributes an important role to complement C3 and its receptor C3aR in basal- and ischemia-induced neurogenesis in young adult mice (Rahpeymai et al., 2006
). The reduction of NeuN positive neurons in APP;C3−/−
mice may be due to a reduction of neurogenesis over the lifespan of C3 deficient mice. However, because the average number of hippocampal CA3 NeuN-positive neurons per section was similar in 12 month-old APP;C3−/−
versus APP mice this seems unlikely. Quantification of DCX positive cells in the granular cell layer (GCL) of the dentate gyrus, the source for new hippocampal neurons, was not possible as the DCX staining in GCL was below detectable levels in the 12 and 17 month-old APP and APP;C3−/−
mouse brain sections (in contrast to DCX positive cell clusters in the SVZ), consistent with earlier reports in APP mice (Jin et al., 2004
In addition, C1q and C3 were recently reported to play a role in CNS synapse elimination as shown by the failure of C1q-deficient and C3-deficient mice to fully refine retinogeniculate connections during postnatal development (Stevens et al., 2007
). Complement-independent mechanisms of synapse elimination may also exist (as suggested by the authors) and may have allowed synapse elimination during development in our APP;C3−/−
mice. Furthermore, initial studies in a glaucoma mouse model by the same group suggest that complement-mediated synapse elimination may become aberrantly reactivated under disease conditions via upregulation of C1q at the synapse leading to synaptic and neuronal degeneration. The presence and possible upregulation of complement C1q in our APP;C3−/−
mice with aging may have contributed to the synapse reduction and neuronal loss we observed at 17 months of age.
In summary, we demonstrate that C3-deficiency in APP mice resulted in increased cerebral Aβ deposition and neuronal loss as well as a shift to a M2 microglial/macrophage response, thereby supporting a beneficial, neuroprotective role of complement C3 in brain. Further studies are underway to further tease apart the various mechanisms by which complement proteins influence neuronal health at different ages.