Our current data suggest that the reaction of complement factors differs between Chandler and 22L scrapie strains. There are multiple distinct prion strains distinguished by biological and biochemical typing, even though the amino acid sequence of the constituent protein is identical. Strains can differ in incubation period, neuropathology, cellular tropism, the size of protease-resistant PrPSc
glycosylation ratios, and PrPSc
conformation (Bruce et al., 1989
; Bessen and Marsh, 1992
; Caughey et al., 1998
; Safar et al., 1998
; Kuczius and Groschup, 1999
; Mahal et al., 2007
). In this study, we used two mouse-adapted scrapie strains, Chandler and 22L. These two strains can be distinguished by biochemical typing, such as PrPSc
glycoform profiles, replication efficiency in cell culture, and conformation (Nishida et al., 2000
; Atarashi et al., 2006
; Mahal et al., 2007
; Sim and Caughey, 2009
; Baron et al., 2011
). In contrast, biological properties of these strains, such as incubation period, distribution of pathological lesions and deposition of PrPSc
, are similar, although the vacuolar lesions in the cerebellum are more prominent in Chandler-infected mice (data not shown). Our study provides evidence that differential reactivity with complement factors is a biological feature that discriminates the Chandler and 22L strains. Accordingly, it will also be of interest to compare the involvement of complement factors in infections with other mouse-adapted scrapie strains, such as Obihiro, G1 and ME7, that have distinct biological properties (i.e., incubation period, distribution of pathological lesions, glial activation).
The reason for the difference in complement factor reactivity between the Chandler and 22L strains remains unclear. Our confocal microscopy data indicate that PrP was colocalized with C1q in Chandler-infected N2a cells, whereas PrP in 22L-infected N2a cells was colocalized with C3. The resolution of confocal microscopy does not allow us to conclude unequivocally that complement factors bind directly to PrP or PrPSc
. However, one of the possible explanations for different complement reactivities of Chandler and 22L PrPSc
is that they directly bind to C1q and C3 with different relative affinities. Another possibility is that these strains have different affinities for inhibitors of C5 convertase and MAC formation, such as C4b binding protein, clusterin, CD59 and fibronectin (Speth et al., 2008
), resulting in differences in MAC formation on Chandler and 22L-infected N2a cells. Recombinant PrP, fibrils, oligomers and purified PrPSc
directly bind C1q in vitro and induce complement activation (Blanquet-Grossard et al., 2005
; Dumestre-Perard et al., 2007
; Mitchell et al, 2007
; Sim et al., 2007
; Sjoberg et al., 2008
). Moreover Veerhuis et al., (2005)
reported that C1q enhanced PrP-peptide fibril formation. C4b binding protein also binds recombinant PrP in vitro (Sjoberg et al., 2008
). However, further study is needed to demonstrate whether pathogenic forms of PrP bind complement factors and/or their inhibitors in prion infections in vivo and in cultured cells.
We didn’t detect colocalization of C3 and PrP in Chandler-infected N2a cells, which seems to be conflicting with respect to involvement of C3 for the Chandler strain in translocation of phosphatidylserine in N2a cells. We suggest two possibilities for the reason. One is that C3 may be involved in the process of MAC formation through classical and/or lectin pathways without directly binding to PrP/PrPSc. Another possibility is that C3 may bind PrP only in the absence of C1q because of the former’s lower affinity to PrP/PrPSc. Extensive additional experiments would be required to discriminate between these possibilities.
Annexin V has a high affinity for phosphatidylserine (Koopman et al., 1994
), which is exposed from the inner layer to the outer layer of the plasma membrane in the early stages of cell death by apoptosis and necrosis (Fadok et al., 1992
). In the current study, we found that infected N2a cells were stained with Annexin V after NMS treatment. However, the cells had not progressed to either full-blown apoptosis or necrosis because less than 10% of the cells were stained with PI at 24 h after treatment. In addition, the cells were negative for cleaved caspase-3 at 24 h (data not shown). A similar phenomenon has been reported when human B cells are treated with C3 (Løbner et al., 2009); i.e., the cells were positive for Annexin V, but were negative for cleaved caspase-3. Segmentation of nuclei was not observed. Therefore, our data suggest that complement factors induced translocation of phosphatidylserine, without cell death. The other factors may be required for cell death in prion infections.
The relatively strong deposition of PrPSc in the thalami of Chandler- and 22L-infected mice at the preclinical 90 dpi time point seems likely to account for the mild vacuolation and deposition of either C1q or C3, respectively, in this region. Although we do not have any evidence that these complement factors were activated, there is a possibility that complement activation may occur relatively strongly in the thalamus at this time point, and be involved in neuropathogenesis. However, the functional/clinical consequences thalamic lesions in particular are not clear because by the onset of clinical signs, the accumulation of PrPSc, complement factors, gliosis and vacuolation are more evenly distributed throughout the brain.
The immunohistochemistry data showed widespread distribution of C1q both in Chandler- and 22L-infected mouse brains, although the dorsal part of thalamus in 22L-infected mice lacked C1q immunoreactivity. We suspect that signals stimulating C1q synthesis may not be different between Chandler and 22L infection, but that activation of C1q might be limited in 22L infections. Indeed, C1q in normal mouse serum was not involved in inducing Annexin V positivity in 22L-infected N2a cells. With respect to the immunohistochemistry data, we think that that reactivity of complement factors may be different in vivo as well as in N2a cells.
It is also unclear whether complement activation might work to alleviate or worsen disease because of multifunctionality of complement factors in vivo. Nonetheless, we suggest two possibilities for the roles of complement factors in prion infections from our current data. One possibility is that complement factors facilitate microglial phagocytosis of prion-infected neurons by exposing phosphatidylserine on the cell surface. Although phosphatidylserine itself works as an “eat-me” signal and promotes phagocytosis (Marguet et al., 1999
), it has been reported that binding of C1q and C3b on the cell surface also facilitates phagocytosis. Because phosphatidylserine has been known as a C1q binding molecule (Païdassl et al., 2008), exposure of phosphatidylserine may result in further deposition of C1q, which in turn may accelerate phagocytosis of the target cells. Another possibility for the roles of complement factors is to cause degeneration of prion-infected neurons. Bordin & Whitfield (2003)
showed that C1q induced apoptosis in human fibroblasts. In addition, it has been reported that C1q is involved in removal of excess synapses in development (Stevens et al., 2007
). C3 is reported to induce translocation of phosphatidylserine from the inner to the outer of the plasma membrane in human B cells (Løbner et al., 2009). MAC is composed of C5b, C6, C7, C8 and multiple C9 (C5b-9), which form transmembrane channels on the plasma membrane resulting in lysis by fluid influx into the cells. When the number of the C5b-9 molecules on the target membrane is limited, cell lysis does not occur. However, the C5b-9 molecules in sublytic conditions have a pro-apoptotic effect by mediating cellular signaling pathways (Hughs et al., 2000). MAC formed on the Chandler-infected N2a cells could have been sub-lytic in our experiments because cell lysis was not observed. However, some previous studies have reported that complement factors C1q and C3 have anti-apoptotic and neuroprotective effects as well (Rus et al., 1996
; Dashiell et al., 2000
; Benoit and Tenner, 2011
). Interestingly, Erlich et al. (2010)
suggested that C1q binds small oligomers derived from murine recombinant PrP and inhibits cytotoxic effects of PrP oligomers. It is possible that complement factors have both neurotoxic and neuroprotective effects and that the role of the complement factors may be different depending on the stage of the disease.
In conclusion, our data provide evidence that the reaction of complement factors varies with the prion strains and that complement reactions can induce the translocation of phosphatidylserine in the membrane of prion-infected N2a cells. The roles of complement factors in prion infection might be further elucidated in the future using ex vivo systems such as slice cultures and mixed cultures of neurons and glial cells.