Reduced binding of apoE4 to complement factor H promotes amyloid‐β oligomerization and neuroinflammation

Abstract The APOE4 variant of apolipoprotein E (apoE) is the most prevalent genetic risk allele associated with late‐onset Alzheimer's disease (AD). ApoE interacts with complement regulator factor H (FH), but the role of this interaction in AD pathogenesis is unknown. Here we elucidate the mechanism by which isoform‐specific binding of apoE to FH alters Aβ1‐42‐mediated neurotoxicity and clearance. Flow cytometry and transcriptomic analysis reveal that apoE and FH reduce binding of Aβ1‐42 to complement receptor 3 (CR3) and subsequent phagocytosis by microglia which alters expression of genes involved in AD. Moreover, FH forms complement‐resistant oligomers with apoE/Aβ1‐42 complexes and the formation of these complexes is isoform specific with apoE2 and apoE3 showing higher affinity to FH than apoE4. These FH/apoE complexes reduce Aβ1‐42 oligomerization and toxicity, and colocalize with complement activator C1q deposited on Aβ plaques in the brain. These findings provide an important mechanistic insight into AD pathogenesis and explain how the strongest genetic risk factor for AD predisposes for neuroinflammation in the early stages of the disease pathology.


Introduction
According to current understanding, accumulation of Ab in the brain is caused by increased Ab aggregation and impaired clearance of Ab. The current findings on the association of complement markers with Ab plaques and complement genes with risk of AD highlight the role of complement system in the disease pathogenesis (Lambert et al, 2009). Previous mouse models have demonstrated that the classical pathway (CP) components C1q and C4 contribute to microglia-mediated loss of brain synapses in early AD (Hong et al, 2016). Ab aggregates are known to activate the complement system in the brain, which triggers microglia activation. These activated microglial cells promote neuroinflammation and neurotoxicity and are involved in Ab phagocytosis (Shen & Meri, 2003;Zhang et al, 2011).
Binding of C1q to apolipoprotein E (apoE) has been suggested to either initiate CP activation on surfaces or reduce the activation in solution, implicating that apoE, a major protein related to AD, is important in target recognition (Yin et al, 2019;Vogt et al, 2020). From the three allelic apoE isoforms (apoE2, apoE3, and apoE4), apoE4 is strongly associated with AD, while apoE2 is protective (Saunders et al, 1993). Idiopathic hydrocephalus (iNPH) is a neurodegenerative disease seen in aging population. The frontal cortex biopsy sample taken from these patients at shunt placement shows high frequency of amyloid b (Ab) pathology and markers that correlate with early Alzheimer's disease (AD; Huang et al, 2021a). The apoE-related AD risk also applies to patients with iNPH. ApoE is found abundantly in Ab plaques. Several studies suggest that apoE and Ab form complexes in fluid phase and the stability of this complex formation is isoform-specific being higher with apoE3 compared to apoE4 (Bentley et al, 2002). There is also evidence that apoE influences Ab cellular uptake through competitive binding to cell surface receptors (Verghese et al, 2013). ApoE is secreted as a lipid-free form mainly by astrocytes and lipidated by ATP-binding cassette transporters (Yamazaki et al, 2019). A small portion of apoE, however, is found associated with lipid-poor proteins, from which especially apoE4 is susceptible to aggregation (Hatters et al, 2006). The current understanding is that apoE is involved in Ab clearance, but the mechanisms underlying these effects are not fully known.
ApoE interacts with complement regulator factor H (FH) and reduces complement activation in plasma (Haapasalo et al, 2015). FH is found abundantly in plasma but is also expressed at lower levels in the brain (Huang et al, 2021a). It is essential for keeping complement activation under control and preventing complement attack toward host tissues (Walport, 2001). It acts as a cofactor to factor I in inactivation of C3b to iC3b, which is the ligand for complement receptor CR3, and thereby promotes non-inflammatory clearance of unwanted material by macrophages (Gershov et al, 2000). FH is able to distinguish self-cells from non-self-cells through recognition of host-specific markers such as Nacetylneuraminic acid, which is abundantly expressed on human cell surfaces. It also binds to self-surfaces indirectly via C-reactive protein (CRP; Haapasalo et al, 2015). When bound to surfaceassociated CRP, it inhibits alternative pathway (AP) activation and C3b generation, which can act as an amplification loop through all the three complement activation pathways (Walport, 2001). FH consists of 20 homologous domains, which form an elongated modular structure. The surface recognition sites on FH are located in domains 5-7 and 19-20. Therefore, several known mutations and polymorphisms in FH and anti-FH antibodies directed against these domains are associated with diseases (Jokiranta, 2017). Interestingly, there are some indications that a Y402H polymorphism in domain 7 of FH, which significantly increases the risk to age-related macular degeneration (AMD), could also be associated with AD, although conflicting results on this association have been found in different genetic studies (Zetterberg et al, 2008;Le Fur et al, 2010).
This study was undertaken to provide insight into a potential mechanism by which interactions between FH and apoE work to reduce Ab toxicity, plaque formation, and neuroinflammation, which have been implicated in the pathogenesis of AD. Here we show that interaction between apoE and FH, which is apoE isoform-specific both in vitro and in vivo, alters their concerted action in reducing Ab1-42 oligomerization, interaction with the phagocytic receptor CR3 and Ab1-42 mediated inflammatory responses and cytotoxicity. The unique data obtained from biopsy tissue from living iNPH patients show colocalization of apoE and FH near Ab plaques and complement activator C1q. Consistent with our in vitro findings, the increased expression of complement and AD-associated proteins in the brain of at-risk apoE4 allele carriers provides further evidence of the protective role of apoE-FH interaction in Ab-induced neuroinflammation.

Affinity between apoE and FH is apoE isoform-specific both in vivo and in vitro
To gain insight into the interaction between apoE and FH, we studied binding of FH to apoE in vivo and whether these polymorphisms could affect this interaction. We used cortical brain biopsy samples obtained from apoE genotyped iNPH patients as a tool to understand the Ab induced neuroinflammation in vivo, as 50% of iNPH patients develop lesions of Ab plaques leading eventually to development of AD (Luikku et al, 2019;Appendix Table S1, patient data). The immunohistochemistry analysis revealed colocalization of apoE and FH associated with Ab and on the surroundings of Ab plaques and brain capillaries (Fig EV1A-F). Immunostaining and colocalization analysis showed significantly increased apoE-FH colocalization in the presence of apoE23 when compared to apoE44 (Fig 1A-D). Interestingly, some but not all Ab plaques contained an apoE core ( Fig 1C). In the brain, the lipidation of secreted apoE varies from lipidated to non-lipid or lipid-poor form, whereas the profile and degree of apoE glycosylation varies depending on location. This variation did not, however, affect the apoE isoform specificity of apoE-FH interaction as this finding was confirmed using immunoblotting of HDL particles isolated from plasma of apoE23 and apoE44 carrying iNPH patients (Figs 1E and EV1G). This interaction was apoE specific as the exogenously added FH co-immunoprecipitated with apoE from the HDL. The observation that binding of FH to HDL was significantly inhibited by the N-terminal fragment of apoE2(1-165) but not by apoE4(1-165; Fig 1F) showed that the C-terminal lipid binding domain is not essential for apoE-FH interaction. As expected, binding of immobilized recombinant apoE to FH in vitro was also apoE isoform specific as shown by significantly increased binding of apoE2 and significantly reduced binding of apoE4 to FH ( Fig 1G). As published before, C1q bound all the apoE isoforms equally well (Yin et al, 2019). A similar difference between binding of FH to apoE2 and apoE4 was observed when both E. coli expressed and glycosylated mammalian cell expressed apoE recombinants were used ( Fig 1H). Consistently, apoE2 and apoE3 bound FH with affinities of 2.6 and 1.0 lM in Microscale thermophoresis (MST), respectively, while apoE4 did not interact with full-length FH (Fig 1I  and Appendix Table S2, MST statistics). The N-terminal fragment of apoE2 bound both the wild-type FH402Y and the FH402H AMD risk variant equally well, while a lower affinity binding was observed to FH domains 1-4 ( Fig 1J). ApoE2(1-165), however, did not bind FH domains 19-20 (FH19-20). As expected, apoE2(1-165) bound the recombinant fragments of FH domains 5-7 (FH5-7402Y and FH5-7402H) equally well ( Fig 1K). Together, these data suggest that binding of apoE to FH is apoE isoform-specific both in vivo and in vitro with apoE4 displaying diminished FH binding capacity. The apoE binding site in FH is located within domains 5-7, similarly as published before (Haapasalo et al, 2015), but it may expand to domain 4 while the N-terminal fragment of apoE is sufficient for apoE-FH interaction. More specifically, a molecular model of the interaction complex of apoE with FH5-7 based on crosslinking mass spectrometry and molecular docking combined with molecular dynamics simulations suggests the N-terminal LDLR receptor binding region (136-150) of apoE2 is the major binding site for FH5-7 (Figs 1L and EV2A and B; Dataset EV1; Chen et al, 2021). Also, the structural model suggests that the Y402 is outside the binding interface, in agreement with the observation that mutation Y402H does not affect the binding affinity.
ApoE isoform-specific binding of FH to apoE/Ab1-42 complexes reduces Ab aggregation It has been suggested that native apoE forms stable apoE/Ab complexes in an isoform-specific manner and thereby affects Ab clearance (Bilousova et al, 2019). In the fluorescent images of iNPH biopsy samples we detected colocalization of apoE and FH with small Ab plaques but not with large Ab plaques (Fig EV1A-C) suggesting that FH could affect the stability of apoE/Ab complexes. To study apoE/Ab complex formation in solution, we fractionated the proteins and protein complexes by size exclusion chromatography and analyzed the fractions by ELISA. We observed that both apoE2 and apoE4 eluted in association with Ab1-42 and FH, while FH without apoE eluted mainly as a single molecule (Figs 2A and EV3A). In SDS gels, the stability of apoE/Ab was isoform-specific ranging from apoE2 being most stable via apoE3 to apoE4 being less stable (Figs 2B and EV3B and C; Appendix Table S3). Consistent with the observed apoE isoform-specific interactions between apoE and FH, FH formed stable complexes with apoE2 and apoE3 but not with apoE4 or Ab1-42 alone irrespective of the presence of Ab1-42 in the complex indicating that FH binds apoE on the complex.
To verify the oligomeric state of Ab1-42 and stoichiometry of apoE2/Ab1-42 and apoE3/Ab1-42 complexes, we performed single molecule total internal reflection fluorescence (TIRF) imaging using fluorescently tagged Ab1-42 and apoE2 in the presence and absence of FH. Distinct foci of Ab1-42 and apoE were detected in the green and red channels respectively. The majority of foci were colocalized (Fig 2C and D) consistent with apoE2/Ab1-42 complexes except when FH was also included which significantly decreased the proportion of colocalized foci, consistent with FH disruption of the complex formation . Reduction in the number of fluorophores/foci that measures the stoichiometry of the apoE/Ab1-42 complex was more striking on apoE2/Ab1-42 complexes than apoE3/Ab1-42 complexes in the presence of FH. Molecular counting analysis of foci using step-wise photobleaching ( Fig EV4) revealed that both apoE2/Ab1-42 and apoE3/Ab1-42 from 10 to nearly 1,000 fluorescent molecules (Fig 2E), dropping to only a few 10s apoE2/Ab1-42 complexes in the presence of FH. These data indicate that FH binds to apoE on apoE/Ab1-42 complex in an isoform-specific manner (apoE2 > apoE3 > apoE4). Binding of FH to apoE restricts the size of soluble Ab aggregates and may thereby facilitate clearance of apoE/Ab1-42 complexes. This finding may explain why toxic Ab aggregates are formed preferentially in the presence of apoE4 (Hatters et al, 2006). FH can be found in the vicinity of phagocytic cells and Ab plaques in vivo and competes for binding with apoE and soluble Ab1-42 to CR3 in vitro Increasing evidence suggests that extracellular accumulation of toxic Ab aggregates is caused by imbalance between Ab production and clearance. ApoE forms a complex with Ab that is efficiently taken up by microglia (Yeh et al, 2016). Cellular uptake of Ab is mediated through various cellular receptors such as the phagocytic receptor CR3 (Doens & Fernandez, 2014). Expression of CR3, however, has been shown to increase rather than reduce Ab accumulation in vivo (Czirr et al, 2017). CR3 has an important role in mediating phagocytosis of iC3b-opsonized particles but it also interacts with various ligands including Ab and FH. Moreover, CR3 is also involved in reducing extracellular Ab degradation and mediates neurotoxic ◀ Figure 1. ApoE-FH interaction is apoE isoform-specific.
Immunofluorescence staining of apoE44 (n = 3) and apoE23 (n = 2) genotyped iNPH patient right frontal cortex biopsy samples (See Appendix Table S1). Two representative microscopy images from six separate images show: A Colocalization of (red) apoE44 and (green) FH in the presence of (white) Ab plaques. Scale bar = 50 lm. B Colocalization of (red) apoE23 and (green) FH around brain capillaries. The blue nuclei were detected using DAPI staining. See also Fig EV1 and Appendix Fig S1. Scale bar = 50 lm. C Colocalization of (red) apoE44 and (green) FH in the presence of (white) Ab plaques from a biopsy sample obtained from an iNPH patient diagnosed with Alzheimer's clinical syndrome (ACS). Colocalization of apoE on Ab is shown with an arrow. The negative staining control is shown. See also Appendix Fig S2. Scale bar = 50 lm. D ApoE-FH colocalization analysis of all detected colocalized foci in six microscope images from the apoE44 (n = 3) and apoE23 (n = 2) genotyped iNPH patient biopsy samples. Six microscope images from Alzheimer's clinical syndrome (ACS) biopsy sample are included in the apoE44 dataset (red dots). E (left above) Quantified intensities of two separate WBs showing presence of endogenous FH on HDL particles isolated from apoE44 (n = 2) and apoE23 (n = 2) genotyped iNPH patients (left below) in one representative membrane (See loading control and apoE staining in Fig EV1G). (right) Co-immunoprecipitation of exogenously added FH with apoE23 from HDL particles. F ApoE2(1-165) specific inhibition of FH binding to HDL-associated apoE coated on ELISA plates. G Binding of FH and C1q to different apoE isoforms. H Binding of FH to non-glycosylated (E. coli expression) and glycosylated (mammalian cell expression) human apoE2, and apoE4 and the negative control BSA. I MST showing binding of NT647-labeled full-length FH to different apoE isoforms, J NT647-labeled apoE2(1-165) fragment to increasing concentrations of FH402Y and FH402H variants and FH domains 1-4 and 19-20. K NT647-labeled recombinant N-terminal apoE2(1-165) to increasing concentrations of FH5-7402Y and FH5-7402H fragments. L Structural model for apoE2(1-165)/FH5-7 complex based on crosslinking mass spectrometry and molecular docking and molecular dynamics simulations. The best model ensemble after molecular dynamics simulation run of 250 ns is presented (apoE yellow, FH5-7 green), apoE LDL receptor (LDLR) binding regions 135-150 shown in gray. The residues at the interface conserved in simulations including K143 of apoE are shown as sticks (Nt, N-terminus; Ct, C-terminus). See also Fig EV2A and B and Dataset EV1.
Data information: Error bars indicate SD values calculated from pooled data (n = 1-3 samples/assay) obtained from repeated experiments (n = 3-4). Statistics was calculated using two-tailed one-way ANOVA supplemented with (D) Tukey's or (F-H) Dunnett's multiple comparison test using SPSS software or (I-K) MO. Affinity Analysis Software (NanoTemper) MST signal is shown as change in the normalized fluorescence (ΔFNorm &) calculated by MO. Affinity Analysis Software. See also statistics in Appendix Table S2. Source data are available online for this figure. effects (Zhang et al, 2011;Doens & Fernandez, 2014;Nissil€ a et al, 2018). Because of the suggested harmful role of Ab-CR3 interaction, we wanted to study whether FH in the presence or absence of apoE could selectively block binding of soluble Ab to CR3. First, we examined whether FH, apoE and Ab are located in the vicinity of phagocytic cells in vivo in the biopsy samples from apoE genotyped iNPH patients. We observed clusters of activated cells (microglia, or peripheral-derived macrophages), as demonstrated by Iba-1 staining, around but did not colocalize with FH coated surfaces (Fig 3A and B) and Ab plaques ( Fig 3B). The apoE44 samples had increased levels of both Ab and Iba-1 (Fig 3C-E). The quantity of FH, however, was the same between these samples ( Fig 3E) Figure 2.
Ó 2023 The Authors EMBO reports 24: e56467 | 2023 although a significant difference between apoE-FH colocalization was detected (Fig 1A-D). Ab plaques frequently colocalized with apoE and contained an apoE protein core. FH did colocalize with apoE and Ab plaques and was also observed between Ab and apoE core as well as on the cells and surfaces surrounding the plaques (Fig EV1A-F). We used genetically uniform U937 cells overexpressing CR3 to study the role of FH and apoE isoforms in Ab binding and phagocytosis. The CR3 receptor consists of one a-chain (CD11b) and one bchain (CD18) from which CD11b interacts with multiple ligands including fibrinogen, ICAM, FH, and the inactivated form of C3b, iC3b. The ligand binding site in CD11b is located in the N-terminal aI-domain that is exposed when the receptor is activated and adopt the extended open conformation (Lamers et al, 2021). In the in vitro flow cytometric analysis, all apoE isoforms bound to CR3, when the receptor was in active extended open conformation, suggesting that apoE interacts with the ligand-binding I-domain of CD11b (Fig 3F-H;Chen et al, 2010). The binding of apoE4 to CR3 was, however, significantly reduced when compared to apoE2. The full-length FH but not the fragments of FH, Ab1-42 or BSA inhibited binding of apoE2 to CR3, indicating competitive binding between apoE2 and FH to CR3 and that apoE2-FH complex (Fig 1) does not interact with the integrin. Ab1-42 also bound to the I-domain of CD11b ( Fig 3I,  Appendix Fig S3A), but the affinity of Ab1-42 to CR3 was lower than the affinity of apoE2, apoE4 or FH, as all of these molecules inhibited Ab1-42 binding to CR3. Inhibition was most significant in the presence of both apoE2 and FH, indicating combined inhibitory effect of these molecules in Ab-CR3 interaction. All these data together indicate that apoE and Ab share at least partially the same binding site on activated CR3 ( Fig 3J) and that the affinity of apoE to CR3 is isoform-specific (apoE2 > apoE4). The increased inhibitory function of the apoE2-FH combination could be due to both FH/apoE2/Ab complex formation in fluid phase (Fig 2A and B) and competitive binding between Ab and apoE or Ab and FH to CR3. Because binding of Ab to CR3 may promote neurotoxic effects (Zhang et al, 2011), the observed antagonizing function of apoE2 and FH may have important anti-inflammatory effects.
FH associated with apoE/Ab1-42 complex increases complement and phagocytosis resistance Activation of the AP is initiated through spontaneous hydrolysis of C3 and therefore, without sufficient down-regulation it can attack our own cells. The hydrolyzed C3, binds factor B, exposing factor B ◀ Figure 2. Binding of FH to apoE/Ab1-42 complexes reduces Ab aggregation.
A ApoE2, Ab1-42 and FH (apoE2/Ab1-42/FH), apoE4, Ab1-42 and FH (apoE4/Ab1-42/FH) and Ab1-42 and FH (Ab1-42/FH) were incubated for 72 h, and the complexes were isolated by size exclusion chromatography. The elution profiles of only FH, only Ab1-42 and only apoE2 and the size exclusion standard proteins are also shown. B Different mixtures of apoE isoforms, FH, FH19-20 and Ab1-42 were incubated for 72 h, and the presence of stable complexes was analyzed by running the sample in Tris-glycine PAGE for (left and middle) 90 or (right) 60 min at 100 V in the presence of 1 x Bolt TM LDS Sample Buffer (Thermo Fisher). Gels were subjected to silver staining (left) or WB using anti-Ab antibody (middle and right). ApoE isoform and incubation time are shown on the top. The presence of each protein is indicated with arrows. Ab1-42 aggregate and the stable complexes formed between apoE, Ab1-42 and FH are shown near the top of the gel, while apoE2/Ab1-42 formed in the absence of FH are shown in several bands below apoE/Ab1-42/FH complexes. See also Fig EV3 for ELISA, anti-FH/anti-apoE dual fluorescence labeling, SDS-PAGE and native PAGE and Appendix Table S3 for band intensities. C Single-molecule TIRF micrograph of Hylite 488 labeled Ab1-42 and Alexa Fluor 568 C5 Maleimide labeled apoE2. Scale bar = 5 lm. D Jitter plot of proportion of colocalized Ab1-42/apoE2 and Ab1-42/apoE3 over multiple fields of view in the presence and absence of FH. Statistics was calculated using Student's t-test from multiple images (n = 12) in each sample. Error bars indicate SD values. E Jitter plot of Ab1-42 foci stoichiometry in number of fluorophores colocalized or not with apoE2 and apoE3 and in the presence and absence of FH. Statistics of multiple images was calculated using two-sided Student's t-test. Statistics was calculated using Student's t-test from all detected colocalized foci in multiple images (n = 12) in each sample. Error bars indicate SD values. See also Fig EV4. Source data are available online for this figure.
▸ Figure 3. FH localizes near phagocytic cells and Ab plaques and competes for binding with apoE and Ab1-42 to CR3.
A, B Immunofluorescence staining of apoE44 (n = 2) and apoE23 (n = 2) genotyped iNPH patient right frontal cortex biopsy samples (See Appendix Table S1) showing localization of FH (green) with Iba-1 stained cells (red) and clustering around cells and brain capillaries. Presence of (white) Ab plaques in apoE44 samples and (arrow) FH in both samples are shown. C-E (C) Iba-1, (D) Ab, and (E) FH intensity analysis was calculated from all detected foci in six microscope images from the apoE44 (n = 2) and apoE23 (n = 2) genotyped iNPH patient biopsy samples. Data information: Error bars indicate SD values calculated from pooled data (n = 1-3 samples/assay) obtained from repeated experiments (n = 3-4). Statistics was calculated using (C-E) Student's t-test or (F-I) two-tailed one-way ANOVA supplemented with Dunnett's multiple comparison test using SPSS software. Source data are available online for this figure. to cleavage by factor D, thus forming C3(H 2 O)Bb that cleaves fluid phase C3 to C3b and C3a. C3b forms deposits on the target surface and when inactivated to iC3b it acts as an opsonin to CR3. C3a is released in fluid phase, which induces inflammation through interaction with C3aR (Walport, 2001). This signaling has been suggested to promote vascular inflammation and blood-brainbarrier (BBB) dysfunction (Bhatia et al, 2021). Ab aggregates activate the complement system in the brain and thus, trigger the development of inflammation (Shen & Meri, 2003). Because we observed that FH forms more stable complexes with apoE2/Ab1-42 than with apoE4/Ab1-42 and reduces formation of large Ab aggregates, we hypothesized that the interaction between apoE2 and FH could be a crucial mechanism to downregulate complement activation on Ab. Consistent with the known immunoreactivity of Ab, the Ab plaques contained complement C1q in vivo (Fig 4A and B) as marker of CP activation and C3b deposition (Baik et al, 2016). Importantly, FH and C1q were colocalized on Ab plaques ( Fig 4A) indicating simultaneous rather than competitive binding of C1q and FH on Ab plaques. This was verified by in vitro ELISAs where C1q showed some, but not significant, reduction in binding of FH to apoE2 and apoE3 ( Fig 4C), while no inhibition of C1q binding by FH to any of the apoE isoforms was detected. As expected, binding of FH, but not C1q, to apoE was isoform-specific (apoE2 > apoE3 > apoE4) providing increased competitive advantage of C1q over FH for binding to apoE4, which triggers complement activation. Importantly, binding of FH to Ab coated wells was only observed in the presence of apoE2 and apoE3 but not in the presence of apoE4 or Ab1-42 alone. This was consistent with the size exclusion chromatography and PAGE experiments ( Figs 2A and B,and EV3A and B) showing that FH does not bind Ab1-42 alone but binding of FH to Ab/apoE is mediated via apoE2 or apoE3.
In the absence of regulation also the C1q initiated CP activation eventually leads to C3b formation and AP activation (Vogt et al, 2020). To further understand the complement inhibitory role of FH on apoE coated Ab plaques, we next analyzed FH activity upon complement activation when bound to apoE2/Ab1-42 and apoE4/Ab1-42. The protein complexes were coupled into latex beads and their ability to activate and inactivate the first steps of AP was analyzed. As expected, all the apoE-containing Ab1-42 complexes that were not associated with FH-activated complement as detected by the release of C3a in the fluid phase ( Fig 4D). However, the presence of FH reduced C3a levels in all samples but significantly in the supernatant of the FH/apoE2/Ab1-42 complex when compared to Ab1-42/FH. FH was functional on all the samples as shown by the presence of the cleavage fragment of the a-chain of C3b, iC3ba, in the immunoblot (Fig 4E; Appendix Table S4). Importantly, a reduction of all C3b fragments was observed especially on FH/apoE2/Ab1-42 indicating inhibition of complement amplification and C3b formation. Together these results evidenced that FH is functional when associated with apoE/Ab1-42 complexes. The increased binding of FH to apoE2/Ab1-42 reduced formation of C3a and deposition of C3b and iC3b while reduced binding of FH to apoE4/Ab1-42 and formation of larger numbers of C3a, C3b, and iC3b indicated induced inflammation and increased CR3 mediated phagocytosis of these Ab complexes (Haapasalo & Meri, 2019).

FH inhibits phagocytosis of apoE/Ab1-42 complexes and thereby reduces Ab1-42-mediated cell toxicity
According to current understanding, Ab phagocytosis may play a less important role in Ab clearance as the majority of extracellular Ab is removed across the BBB (Zhao et al, 2015). This is also consistent with the finding that CSF Ab levels are significantly reduced in AD patients when compared to healthy individuals (Palmqvist et al, 2014). To further understand the role of FH in clearance of apoE/Ab complexes, we investigated whether iC3b and CR3 are ▸ Figure 4. FH colocalizes with Ab-associated C1q in vivo and reduces complement activation, phagocytosis, and toxicity of apoE/Ab1-42 complexes.
A Immunofluorescence staining of iNPH patient biopsy samples (apoE33; See Appendix Table S1, patient data) showing (arrow) colocalization of complement activator, (red) C1q, and complement regulator (green) FH. B (arrow) Colocalization of (green) apoE and (red) C1q on (white) Ab plaques. Colocalization analysis is calculated from colocalized foci in two separate microscope images. C Simultaneous binding of C1q and FH to different apoE isoforms in vitro.    involved in the uptake of complement challenged protein complexes using flow cytometry. Somewhat surprisingly, uptake of apoE/Ab1-42 correlated positively with cell permeability (Fig 4F). This was not caused by overactivation of complement as no correlation was observed between C3a formation and cell permeability. However, when the activated empty and CR3-expressing U937 cells were challenged to apoE/Ab1-42, the presence of FH, even when challenged to complement, showed significant reduction in phagocytosis ( Fig 4G). This observation suggests that formation of iC3b on these complexes has some but a less important effect in phagocytosis than expected and that exaggerated uptake of cytotoxic Ab1-42 is mediated through various receptors (Kam et al, 2013;Czirr et al, 2017). Interestingly, regulation of phagocytosis of Ab1-42 complexes by the concerted action of FH and apoE2 significantly reduced Ab1-42 cytotoxicity ( Fig 4G).

Inhibition of apoE/Ab1-42 phagocytosis by FH alters AD-associated microglia cell responses in vitro
The presence of Ab is known to activate microglial cells. Therefore, we next wanted to understand how microglial cells respond to the presence of apoE and FH by challenging microglial SV40 cells with different apoE isoforms and Ab1-42 in the presence or absence of FH. As expected, Ab1-42 phagocytosis was significantly reduced in the presence of Ab + FH + apoE2, and Ab + FH + apoE3, but not in the presence of FH + apoE4 when compared to the cells incubated only with Ab1-42 ( Fig 5A; Appendix Fig S3B).
The live cell imaging analysis, however, showed that the uptake of Ab1-42 was not completely inhibited in the presence of Ab + FH + apoE2 indicating that apoE and FH direct a controlled uptake and clearance of Ab by microgial cells (Movies EV1 and EV2). The transcriptome analysis of the microglial cells revealed a number of genes that showed differential expression (DE) between cells exposed to different combinations of apoE isoforms and FH upon Ab phagocytosis.  Table S5 and Dataset EV2). On the other hand, those genes upregulated in AD, such as AHNAK (Manavalan et al, 2013) and AEBP1 (Shijo et al, 2018), have different physiological roles and may participate in neurodegeneration. Five of the detected genes (DAAM1, PGBD1, PLXNA1, RBBP7, AKT1) also show similar changes in transcription levels and correlation with Ab pathology in a recent mRNA transcriptomic data from iNPH biopsy samples (Huang et al, 2021a). Microglial cells incubated with FH and apoE4 showed upregulation of genes involved in Ab processing and inflammation (Table 1). For example, PICALM (Moreau et al, 2014) is involved in autophagy and clearance of Ab, BECN1 (Rocchi et al, 2017) aids autophagy and APP processing, AKT1 is important for ROS production (Ahmad et al, 2017), AXL has recently been linked to AD due to its direct role in promoting Ab plaque development through microglial phagocytosis (Huang et al, 2021b), and RICTOR (Lee et al, 2017) is important for protecting the cells from Ab-induced toxicity. Differential expression of some key cellular and molecular mediators of neuroinflammation (Yang, 2019) could also be observed between cells incubated with only Ab and Ab + apoE2 (reduced CXCL8), Ab and Ab + apoE4 (increased TREM2 and CFD), Ab and Ab + apoE2 + FH (increased TLR6 and reduced S100A6). (Yang, 2019) From these neuroinflammatory markers, increased TREM2 expression has previously been shown to correlate with Ab pathology in human biopsies (Huang et al, 2021a). Thus, the differential expression analysis guided identification of new and previously published specific markers linked to pathways ( Fig 5E) that when disrupted or abnormally induced could be linked to the process of neuroinflammation and/or neurodegeneration.
The presence of apoE4 isoform reveals increased complement activity, inflammation and upregulation of AD-associated risk proteins in vivo To have a full scan of local proteomic changes affected by the apoE risk isoform in vivo, the frontal cortex biopsy samples from iNPH patients that were apoE4 carriers (apoE44 or apoE43) and noncarriers (apoE33 or apoE23) were subjected to mass spectrometry (Fig 6, Dataset EV3 and Fig EV5). Analysis of the total proteome and comparison between the apoE4 carriers and non-carriers revealed up-or downregulation of several proteins, from which three (PTPRF, TRA2A, and NISCH) correlated very well with the in vitro ▸ Figure 5. 488-Ab1-42 phagocytosis and microglial transcriptomics in the presence of FH and apoE isoforms.
A 488-Ab1-42 phagocytosis in the presence of FH and apoE isoform. Flow cytometry data are presented as mean fluorescence intensity of the cell population of the sample (See Appendix Fig S3B for gating and histograms). B Heat map of the genes that were up-or downregulated between Ab + apoE2 + FH and Ab + apoE4 + FH or compared to Ab + buffer (See statistics in Dataset EV2).
The fold change is calculated between apoE2 + FH and apoE4 + FH. The trimmed mean of M values (TMM-normalized) of mRNA transcription levels from the RNAseq data from two individual samples obtained from repeated experiments (n = 3) is presented with different combinations of FH, apoE and Ab1-42 proteins. Heat map was created by heatmapper (Babicki et al, 2016). C Volcano plot of up-or downregulated genes between Ab + apoE2 + FH and Ab + apoE4 + FH. D Principle Component Analysis (PCA) score plot showing the relationship between transcript expression profiles from FH + apoE2 and FH + apoE4 samples (See also Table 1 and Appendix Table S5). E Top pathways identified by IPA analysis (Qiagen) within the differentially expressed genes between Ab + apoE2 + FH and Ab + apoE4 + FH.
Data information: Statistical significances were calculated using two-tailed one-way ANOVA supplemented with Dunnett's multiple comparison tests using SPSS software. Error bars indicate SD values calculated from pooled data (n = 1-3 samples/assay) obtained from four times repeated experiments. Source data are available online for this figure. mRNA data (Figs 5B and 6A and B;Dataset EV2). From these, upregulation of PTPRF is involved in cell adhesion pathway upon response to complement C3a (Jiang et al, 2021). PTPRF and DCAF7 also correlate with Ab pathology in a recent iNPH transcriptomic data indicating a major role of these proteins in apoE isoform and complement-dependent inflammatory response (Huang et al, 2021a). Screening of the MS1 and MS2 scans revealed three key inflammatory markers (CDK5, S100B, and S100A) from which S100B was significantly increased in apoE33/23 samples (Fig 6C; Yang, 2019). Interestingly, S100B is a proinflammatory protein that has been shown to suppress Ab aggregation and toxicity (Cristovao et al, 2018). Four abundant complement activation markers that were detected showed increased levels in apoE44 samples ( Fig 6D). Importantly, these markers did not show any correlation with Ab pathology suggesting that the Ab triggered complement-mediated inflammatory response is apoE isoform-dependent and thereby supports very well our hypothesis on the role of FH in reducing Abmediated inflammation. ▸ Figure 6. Complement activation markers and proteins that correlate with the presence apoE4 risk allele.
A Top 9 proteins that correlate with the presence of apoE4 risk allele in iNPH biopsies between apoE44 or apoE43 (n = 8) and apoE33 or apoE23 (n = 9) carriers (See Appendix Table S1 for patient data and all DE proteins in Dataset EV3). (arrow) Two of the proteins also showed differential expression in vitro (Fig 5B) B Comparison of mRNA transcripts (two individual samples obtained from three times repeated experiments) and MS1 intensities (from 8 apoE44/apoE43 and 9 apoE33/23 carriers) between genes or proteins that showed differential expression in vitro ( Fig 5B) and in vivo. C Analysis of the key AD neuroinflammatory markers obtained from the detected MS1 spectra between apoE44 or apoE43 (n = 8) and apoE33 or apoE23 (n = 9) carriers. D (left) Combined analysis of complement activation markers C3, C4, CFH, and Clusterin obtained from the detected MS1 spectra between samples from apoE44 (n = 3), apoE43 (n = 5), apoE33 (n = 7) and apoE23 (n = 2) carriers (four markers/patient). (center) Combined analysis of complement activation markers C3, C4, CFH and Clusterin obtained from the detected MS1 spectra between samples that were positive (n = 10) or negative (n = 7) for Ab pathology (four markers/patient).
(right) Expression levels of Phosphodiesterase A control, showing similar expression levels in frontal cortex, between samples from apoE44 (n = 3), apoE43 (n = 5), apoE33 (n = 7) and apoE23 (n = 2) carriers The data were normalized against neutral apoE33 samples with no apoE association. E Schematic presentation of the role of isoform-specific binding of apoE to FH in resolution of Ab-mediated inflammation that is impaired in the presence of the ADassociated apoE4 isoform as suggested by the in vitro and in vivo findings.
Data information: (A and B) Fold changes (Log2fc) and P-values (shown) calculated using the DEP R package (Zhang et al, 2018). (C and D) Significances were calculated using two-tailed one-way ANOVA supplemented with Dunnett's multiple comparison tests using SPSS software. Source data are available online for this figure.

Discussion
The crucial role of FH in preventing complement attack toward host cells is exemplified by the mutations in FH found in atypical hemolytic uremic syndrome (aHUS), where lack of AP regulation leads to endothelial damage and microvascular thrombosis (Jokiranta, 2017). Because FH is located in fluid phase, it can easily reach areas where complement activation is exacerbated, such as Ab plaques. The results of this work provide evidence on the concerted role of FH and apoE in reducing Ab oligomerization and signs of neuroinflammation as these are promoted in the presence of the AD-associated apoE4 isoform. Our data from both in vitro and in vivo analysis suggest that this mechanism may affect the early stage of amyloidogenesis through inhibition of Ab toxicity, by reducing Ab interaction with cells and phagocytosis of toxic Ab (Step 1 in Fig 6E), by reducing Ab oligomerization, and by forming complement resistant FH/apoE/Ab complexes (Step 2 in Fig 6E). FH colocalizes with C1q on apoE-coated surfaces (Step 3 in Fig 6E), suggesting yet another mechanism for target discrimination by FH. Here, high competitiveness of C1q and low competitiveness of FH to apoE4 predisposes the surface to C1q-mediated complement activation. Finally, colocalization of apoE and FH on surfaces and small capillaries in the vicinity of Ab plaques suggests that FH-mediated complement regulation is involved in apoE isoform-specific protection of BBB breakdown (Bell et al, 2012). As FH was abundantly detected around capillaries some of the detected FH in brain parenchyma may have been originated from circulation as leakage of blood-derived proteins have been detected in apoE4 AD post-mortem brain tissues (Zipser et al, 2007). Importantly, all these events are affected by the apoE isoforms, which therefore links FH to the most significant genetic risk factor for AD. FH is the main molecule keeping a delicate balance between complement activation and inhibition. Any disturbance in this balance, such as failure in binding of FH to apoE4 suggested in this study, will eventually lead to inflammation. Our immunohistochemistry and MS analysis using iNPH biopsy samples from living patients correlated well with our in vitro data and revealed a restricted set of novel and recently published molecules that may play an important role in AD pathogenesis. As earlier described, these samples represent early AD pathology and correlate well (but not completely) with mouse in vivo transcriptomic data but not with data obtained from human post-mortem biopsy samples from AD patients (Huang et al, 2021a). In this regard, our data do not include any artifact related to post-mortem changes or differences between species. Under favorable conditions, Ab monomers can form non-fibrillar and fibrillar aggregates both in the extracellular space and intracellularly. Because of the different structure between Ab monomers and fibrillar Ab, the soluble monomers are more easily cleared by phagocytosis, action of secretases or delivery through the BBB (Zhao et al, 2015). In contrast, fibrillar aggregates accumulate, activate the complement system and induce inflammation. Our finding that FH forms complement-resistant complexes especially with apoE2/Ab1-42, which have significantly smaller size than apoE/ Ab1-42 complexes alone, suggests a role of this interaction in inhibiting Ab-induced complement-mediated inflammation and Ab aggregation. Based on the structural MS crosslinking data FH binds to apoE receptor binding site while the residue 402 in FH is not located near this apoE-FH interaction site. Therefore, it is unlikely that the AMD-associated 402H isoform would affect the ability of FH to reduce Ab/apoE oligomerization. While binding of apoE to TREM2 has been shown to facilitate uptake of Ab (Yeh et al, 2016), we show here that apoE and FH both inhibit binding of soluble Ab1-42 to activated CR3 and reduce, but does not completely inhibit, uptake of Ab1-42 by phagocytic cells. Because we observed reduced uptake of Ab1-42 also independently of CR3, it is possible that apoE and FH direct a controlled uptake of Ab through receptors mediating non-inflammatory signals such as TREM2. Interestingly, we observed differential gene expression between microglial cells incubated with FH + apoE2 and FH + apoE4, indicating differential signaling by apoE variants, as suggested by others (Huang et al, 2019). Importantly, when binding to its ligands, CR3 is capable of triggering Ab-induced microglial activation and can potentially constitute another mechanism of Ab neurotoxicity (Zhang et al, 2011). Furthermore, it has been suggested that CR3 may be a potential therapeutic target for the treatment of AD, as knocking out of CR3 has been shown to decrease Ab deposits in the brain of APP-transgenic mice and increase extracellular Ab degradation by microglia (Czirr et al, 2017).
There is a lack of understanding of how exactly Ab is accumulated and cleared in the brain, but the role of microglia in Ab clearance is less important than previously suggested, as 70% of extracellular Ab is cleared by the BBB (Iliff et al, 2012). It is possible that the acidic environment in phagolysosomes may induce formation of Ab fibrils that are compacted into dense-core 'indigestible' material and therefore difficult to phagocytose (Huang et al, 2021b). Our study shows that FH and apoE2 reduce Ab1-42 phagocytosis along with a significant reduction in cell permeability. In the brain, apoE has an important role in maintaining lipid transport and membrane repairing. It has been suggested that Ab toxicity toward cells may be due to its ability to modulate lipid membrane function (Legleiter et al, 2011). Therefore, controlled uptake of Ab to cells by apoE and FH could protect the cells from the direct toxic effect of Ab.
Binding of C1q to apoE on surfaces via the globular domains triggers CP activation (Vogt et al, 2020). We show here that binding of apoE to FH is isoform specific, but not to C1q. This indicates that apoE has a similar physiological function as CRP in acting locally at sites of inflammation to recruit FH and limit complement activation. In plasma, binding of C1q to CRP induces complement activation and C3b formation on damaged cells. Here FH is known to bind CRP, which blocks the AP activation and inflammation caused by the CP complement attack (Mihlan et al, 2009). Importantly, formation of C3b through C1q triggered complement activation is not sufficient for CR3-mediated clearance by microglial cells as factor Imediated cleavage of C3b in the presence of a cofactor is necessity to form the CR3 ligand iC3b. It has been shown that C1q and Ab play a role in synaptic pruning in early AD that is dependent on CR3-mediated microglial engulfment of synapses (Hong et al, 2016). The reduced binding of FH to apoE4, shown in this study, could play a major role in the formation of iC3b that mediates CR3dependent phagocytosis of synapses. In conclusion, our studies have provided novel insights into the mechanism of AD pathogenesis, which may have implications in facilitating studies aiming to find molecular targets for drug discovery to prevent neurodegeneration.

Patient material
Right frontal Cortical Biopsy samples and plasma were obtained from patients with idiopathic normal pressure hydrocephalus (iNPH). Informed consent was obtained from all subjects in accordance with the Declaration of Helsinki and the Kuopio University Hospital Research Ethics Board approval (276/2016(276/ , 8-Sep-2020. Biopsy samples from patients with iNPH were taken prior to the insertion of the ventricular catheter of the cerebrospinal fluid (CSF) shunt as previously described (Koivisto et al, 2016).

Isolation of HDL from plasma and detection of FH
HDL was isolated from plasma of iNPH patients with apoE23 (n = 2) or apoE44 (n = 2) genotype by sequential flotation in an ultracentrifugation using potassium-bromide for density adjustment as previously described (Syed et al, 2021). The methodology of the preparations was as follows: Plasma VLDL and LDL (d < 1.063 g/ml) and total HDL (1.063 < d < 1.210 g/ml). Isolated HDL was dialyzed against phosphate-buffered saline (PBS pH 7.4), visualized by SDS-PAGE, and checked for purity (Fig EV1G). Protein concentrations of the samples were measured with the Bicinchoninic acid assay (BCA; Pierce Biotechnology, Rockford, IL, USA). For Western Blotting (WB), 50 lg of each sample was run on gel as previously described under reducing conditions and transferred (iBlot, Thermo Fischer Scientific) to nitrocellulose membrane (iBlot TM 2 Transfer Stack, Invitrogen) and the membrane was blocked with 3% fat-free milk in PBS for 2 h at room temperature. To detect FH from HDL fractions, the membrane was incubated with 1:2,000 diluted goat anti-factor H (Cat# 341276, Millipore) in 0.3% fat-free milk in PBS for 1 h at room temperature, washed three times with PBS, and incubated with 1:5,000 diluted IRDyeâ 800CW labeled anti-Goat IgG, (Cat# 926-32213, LI-COR) for 1 h at room temperature. The membrane was washed with PBS and imaged using Odysseyâ CLx Imaging System (LI-COR).

Co-immunoprecipitation
ApoE-genotyped HDL particles isolated from one iNPH patient (apoE23) were mixed with FH (Cat#A137, Complement technology) and diluted in PBS so that the final concentration of HDL particles is 0.5 mg/ml (total protein concentration) and that of FH is 0.04 mg/ml. This mixture was incubated at 37°C for 30 min. Meanwhile, mouse monoclonal anti-FH antibody, 3D11 (Fontaine et al, 1989; or PBS as a negative control) was bound to protein G-coupled Dynabeads (Cat# 10003D, ThermoFisher) by incubating them at 37°C for 10 min with rotation. After one wash with PBS, the beads were incubated with the preincubated HDL-FH sample at 37°C for 30 min with rotation. The beads were then washed by incubating them with PBS plus 0.05% Tween 20 at 37°C for 45 min with rotation to dissociate lipids and proteins comprising HDL particles from apoE that were bound to FH. After one more quick wash with PBS plus 0.05% Tween 20, proteins were eluted from the beads by 50 mM glycine pH 2.7 and immediately neutralized by adding 2 M Tris-HCl pH 7.5. The eluted sample was mixed with Bolt LDS Sample Buffer and Bolt Sample Reducing Agent (ThermoFisher), incubated for 5 min at 95°C and run on PAGE gels (Mini-PROTEAN TGX Stain-Free Protein Gels, 4-20%, Bio-Rad, 60 min, 140 V) in TGS buffer (Bio-Rad). Proteins were transferred into nitrocellulose membranes, and the membrane was blocked with 3% fat-free milk in PBS for 2 h at room temperature. After one wash with PBS, the membrane was incubated with 0.3% fat-free milk in PBS containing 1:10,000 diluted goat polyclonal anti-FH antibody (Millipore) and 1:5,000 diluted rabbit polyclonal anti-apoE antibody (kind gift from Dr. Matti Jauhiainen. The antibody was raised in New Zealand White rabbit) for 1 h at room temperature. The membrane was then washed three times with PBS and incubated with 0.3% fat-free milk in PBS containing 1:5,000 diluted IRDye 800CW donkey anti-goat IgG secondary antibody (Cat# 926-32214, LI-COR) and 1:5,000 diluted IRDye 680RD donkey anti-rabbit IgG secondary antibody (Cat# 926-68073, LI-COR) for 1 h at room temperature. After three washes with PBS, the presence of FH and apoE was detected by using Odyssey CLx imaging system (LI-COR).
Binding of FH to HDL and competition with apoE2(1-165) and apoE4(1-165) To detect the binding of FH to HDL and assess the competition with apoE isoforms, apoE2 (1-165) and apoE4 (1-165), HDL was coated on 96-well plates (SpectraPlate-96 HB, PerkinElmer) at concentration of 20 lg/ml in 50 mM bicarbonate buffer pH 9.6 overnight at +4°C. The wells were blocked with 3% fatty acid-free BSA (Cat#6156, Biowest) in PBS for 2 h at room temperature. After one wash with PBS 20 nM FH (Complement technology) was incubated on plate with 1:3 dilution series of apoE2(1-165) and apoE4(1-165) starting at 200 nM concentration in 0.3% BSA/PBS for 1 h 30 min at 37°C. After wells were washed for three times with PBS, FH was detected with 1:2,000 diluted goat anti-Factor H (Millipore) in 0.3% BSA/PBS by incubating for 1 h at 37°C. Next, wells were washed three times with PBS and incubated with 1:2,000 dilution of HRP conjugated anti-goat IgG antibody (Cat# 705-035-147, Jackson ImmunoResearch Laboratories) in 0.3% BSA/PBS at 37°C for 1 h. After three washes with PBS, o-phenylenediamine dihydrochloride (OPD) substrate (Cat# 34006, Thermo Scientific) was added according to manufacturer instructions. The reaction was stopped with 0.5 M H 2 SO 4 and absorbance was measured at 492 nm.

CR3-receptor binding assay
In order to understand how the molecules of interest bind CR3, we induced bent inactive and extended open active conformations of CR3 by treating the U937 cells with Hank's balanced salt solution (HBSS) without Ca 2+ and Mg 2+ (Gibco) and 5 mM EDTA, or with HBSS, 10 mM HEPES, 2 mM EGTA and 0.5 mM MnCl 2 , respectively (Chen et al, 2010). To study binding of apoE2 and apoE4 to CR3, apoE was labeled by DyLight TM 405 (Thermo Fisher Scientific) according to manufacturer's instructions. Preincubation of Dy-Light TM 405-labeled apoE (degree of labeling 0.25 moles dye per mole protein) with FH, Ab1-42 (Cat#AS-20276, Anaspec Inc.), FH5-7, FH19-20 or molecular biology grade bovine serum albumin (BSA; New England BioLabs) or without any of them was carried out in 96-well round-bottom plates in 5 mM EDTA-or 1 mM Mn 2+containing buffer (described above) for 15 min at room temperature. Then 1.4 × 10 5 U937 or U937-CR3 cells that were prewashed with the corresponding buffer were added to wells. The final concentration for each preincubated molecule was adjusted to 300 nM. The mixture was incubated for 45 min at 4°C in the dark with gentle shaking. To study binding of HiLyte TM Fluor 488-labeled Ab1-42 (Cat#AS-60479-01, AnaSpec Inc.) to CR3, first 645 nM apoE2 and apoE4 with or without 645 nM FH, only 645 nM FH, only 645 nM FH19-20 or only 645 nM BSA were preincubated in EDTAor Mn 2+ -containing buffer (described above) for 15 min at 21°C. Next, 1.4 × 10 5 of U937 or U937-CR3 cells were pre-washed with the corresponding buffer, added to wells, and incubated for another 15 min on ice. After adding 4.4 lM of HiLyte TM Fluor 488-labeled Ab1-42, the mixture was incubated for 45 min at 4°C in the dark with gentle shaking. After incubation, the wells were washed with corresponding buffer once and fixed with 1% (v/v) paraformaldehyde. Next, cells were run to BD LSRFortessa flow cytometer and analyzed using FlowJo V10 software (FlowJo, LLC).

Microglial phagocytosis and mRNA sequencing
Preincubation of 1.4 lM of apoE2, apoE3, or apoE4 with or without 650 nM FH (Complement Technology, Inc.) was performed in 96well round bottom plates (Thermo Fisher Scientific) for 30 min on ice. HiLyte488 TM Fluor-labeled Ab1-42 was incubated in the dark at room temperature in PBS for 72 h and then mixed with microglial cells (2.5 × 10 6 cells/ml). The suspension was immediately pipetted into the wells containing the preincubated mixture of apoE and FH (final concentration of Ab: 2 lM, final amount: 8.7 × 10 4 microglial cells per well). Samples were further incubated for 1 h at 37°C with gentle shaking in RPMI-1640 media (Gibco) supplemented with 0.05% human serum albumin (HSA, Sigma). Samples were divided for the mRNA sequence analysis and for the flow cytometric analysis. For the mRNA sequence analysis, 3 × 10 4 cells were stabilized by RNAlater (Thermo Fisher). RNA sequencing method was designed based on the Drop-seq protocol and carried out as described earlier (Nissil€ a et al, 2018). For flow cytometry, the phagocytosis was stopped by adding and washing the samples with ice-cold RPMI-1640 with 0.05% HSA. Samples were fixed with 1% (v/v) paraformaldehyde (Thermo Fisher) in RPMI-1640 with 0.05% HSA. Next, cells were run to BD LSR Fortessa flow cytometer and analyzed using FlowJo V10 software (FlowJo, LLC).

Single-molecules TIRF microscopy
For stoichiometric studies, Cys thiols in apoE2 were labeled with Maleimide C5 Alexa568 reagent according to manufacturer's instructions (Thermo Fisher Scientific). The degree of labeling was 1 as determined by protein concentrations using absorbance at 280 nm and dye concentrations using absorbance at 579 nm by a Nanodrop ND-1000 spectrophotometer. Protein mixture of 1 lM Maleimide C5 Alexa568-apoE2, 18.5 lM HiLyte 488-Ab1-42 with or without 1.7 lM FH and was incubated for 72 h at room temperature in the presence of 0.14 mM DTT in Protein LoBindâ Tubes (Eppendorf). Imaging was performed on a bespoke single-molecule TIRF microscope constructed around a Nikon Ti-E microscope body, using Obis 488 and 561 nm lasers set to 20 mW, beam expanded to fill the field of view of Photometrics Evolve 512 at 100 nm/pixel with a 100× Nikon 1.49 NA TIRF objective lens. Pre-incubated solutions of labeled Ab1-42, apoE2 and FH were imaged in simple flow cells constructed from glass slides, plasma-cleaned coverslips, and double-sided tape, coated with 5 lg/ml of anti-Ab mAb (clone H31L21, Invitrogen). Multiple fields of view and samples were imaged to generate > 10,000 foci tracks over 1,000 frames at 50 ms/ frame exposure time. Data were analyzed using bespoke MATLAB software  to track and quantify the intensity of foci as a function of time. Briefly, centroids are determined using iterative Gaussian masking and intensity calculated using the summed intensity inside a foci corrected for the local background in a small square region of interest around the foci. Foci were accepted if their signal-to-noise ratio was above 0.4 and lasted longer than three frames. Foci were deemed colocalized if their overlap integral was over 0.75. Stoichiometries were determined by first calculating the intensity of single dyes using photobleaching analysis to use only single fluorophores (Fig EV4A and B).
Step-wise intensity traces found within the first 10 frames (Fig EV4C) were photobleach-corrected using a linear regression of the first 4 points, which we have shown is equivalent to full exponential fitting (Shashkova et al, 2021). Initial intensities were divided by characteristic intensity to obtain stoichiometries.

Oligomerization studies
Different combinations of 3 lM apoE2/3/4, 3 lM FH or 3 lM FH fragments (5-7 or 19-20) were incubated with and without 28 lM Ab1-42 (AnaSpec Inc.) for 72 h at room temperature in PBS in the presence of 0.14 mM DTT in Protein LoBindâ Tubes (Eppendorf). To separate protein complexes, complexes and monomers, the samples were run through gel filtration column (Superdex 200 10/300 GL column, Global Life Sciences Solutions LLC) at 0.5 ml/min flow rate in PBS. Fractions of 300 ll were collected, and the fractions were stored immediately at À80°C until further analysis. The peaks from size exclusion chromatography were analyzed by ELISA to verify the presence or absence of each protein in the complexes (Fig EV3A). The size of the complexes and single molecules was estimated by running the Bio-Rad Gel Filtrations Standard (Cat#1511901) using the same protocol. For PAGE analysis, the protein mixtures were mixed with Bolt LDS Sample Buffer (Thermo Fisher Scientific) or 2 × native sample buffer (1×: 62.5 mM Tris-HCl pH 7.4, 25% glycerol, 1% bromophenol blue), run on PAGE gels (Mini-PROTEAN TGX, 4-20%, Bio-Rad, 60-90 min, 100 V) in Tris-glycine buffer or TGS buffer (Bio-Rad) and detected by silver staining or WB. For WB the proteins were transferred into nitrocellulose membranes, and the membrane was blocked with 3% fat-free milk in PBS for 2 h at room temperature. Next, the membrane was incubated with 1:1,000 diluted rabbit anti-Ab mAb (clone H31L21, Invitrogen) in 0.3% fat-free milk in PBS for 1 h at room temperature, washed three times with PBS and incubated with 1:5,000 diluted IRDye 800CW-conjugated goat anti-rabbit IgG (LI-COR) for 1 h at room temperature. After washing the presence of Ab complexes was detected using Odysseyâ CLx imaging system (LI-COR).

Complement activation and inhibition
To couple protein complexes into latex beads the protein mixtures of 3 lM apoE2, apoE3, or apoE4, 3 lM FH or 3 lM BSA were incubated with and without 30 lM HiLyte TM Fluor 488-labeled Ab1-42 for 72 h at room temperature in PBS in the presence of 0.14 mM DTT. Twenty microliters of these samples were coupled to 56 mg of carboxyl latex beads (Thermo Fisher, 4% w/v, 2 lm diameter, C37278). Before coupling the beads were washed twice with sterile MES-buffer (0.025 M (2-(N-morpholino)ethanesulfonic acid), pH 6.0) and centrifuged between washes at 3,000 g for 20 min. The latex bead-protein mixtures were incubated overnight with gentle mixing at room temperature covered from light. Next, beads were centrifuged at 3,000 g 20 min, and the coupling efficiency was calculated by measuring the ratios between A280 in the supernatant and the original sample using NanoDrop 1000 (Thermo Scientific). Coupling efficiency was above 80% in each sample. Next, coupled beads were washed three times with PBS and resuspended in 50 ll of PBS. To study complement activation capacity of the coupled beads, 4 ll of the bead mixtures were incubated in the presence of 0.75 lM C3 (Cat#A113, Complement technology), 0.75 lM factor B (Cat#A136, Complement technology), 0.075 lM factor D (Cat#A135, Complement technology) and 0.375 lM factor I (Cat#A138, Complement technology) in Ca 2+ /Mg 2+ free Dulbecco's phosphate-buffered saline (D-PBS) with 1 mM of MgCl 2 (Mg-DPBS) for 60 min at 37°C. After incubation, the beads were centrifuged at 3,000 g for 20 min. The beads were again washed twice with PBS, resuspended to 20 ll PBS and stored at 4°C for phagocytosis assay and iC3b WB. C3a was measured from 1:10 diluted supernatant by following the manufacturer's instructions (MicroVue C3a Plus, Quidel). Formation of iC3b on the washed beads was detected by SDS-PAGE and WB. First, 6.5 ll of beads were incubated with 2.5 ll Bolt TM LDS Sample Buffer and 1 ll Bolt TM Sample Reducing Agent (Thermo Fisher) for 10 min at 95°C. Next, the samples were centrifuged at 3,000 g for 20 min, and 5 ll of supernatant was run on PAGE gels (Mini-PROTEAN TGX, 4-20%, Bio-Rad, 60-90 min, 100 V) in TGS buffer (Bio-Rad) including 100 ng of control C3, C3b (Cat#A114, Complement technology) and iC3b (Cat#A115, Complement technology) under reducing conditions. The proteins were transferred into nitrocellulose membranes, and the membrane was blocked with 3% fat-free milk in PBS for 2 h at room temperature. To detect C3 cleavage fragments, the membrane was incubated with 1:130 diluted rabbit anti-C3c antibody (Cat# OSAP 14/15, Behring) in 0.3% fat-free milk in PBS for 1 h at room temperature, washed three times with PBS, and incubated with 1:5,000 diluted IRDye 800CW-conjugated goat antirabbit IgG (LI-COR) for 1 h at room temperature. After three washes the presence of C3 cleavage fragments was detected by using Odyssey â CLx imaging system (LI-COR).

Phagocytosis of beads coated with protein complexes
U937 cells were differentiated to macrophages for 72 h using 150 nM of PMA, washed with DPBS (Gibco) and incubated for 10 min at 37°C with cell stripper (Corning). Detached cells were removed by resuspending the cells carefully in RPMI-1640 with 0.05% HSA. After centrifugation for 10 min at 300 g, the cells were resuspended at a density of 5 × 10 6 cells/ml, and the presence of 95% or more live cells were calculated using trypan blue (Bio-Rad). Next, 1 lg/ml of DAPI was added to the cells, and 10 ll of beads incubated with complement components were mixed with 40 ll of these cells. For compensation cells without DAPI were incubated with 1.2 lM HiLyte TM Fluor 488-labeled Ab1-42, and cells with DAPI were incubated with 1.2 lM of non-labeled Ab1-42. Also, beads coated with only HiLyte TM Fluor 488-labeled Ab1-42 without complement deposition were subjected to the assay to measure the effect of C3b and iC3b deposition on phagocytosis. Samples were incubated for 1 h at 37°C with 5% CO 2 atmosphere with mild shaking. Phagocytosis was stopped with 300 ll of cold RPMI-1640 with 0.05% HSA, and cells were centrifuged as above and fixed with 200 ll of 1% formaldehyde in RPMI-1640 with 0.05% HSA. Eighty microliters of cells were diluted into 200 ll of ice-cold PBS, run on FACS flow cytometer (2,000-10,000 cells/sample), and analyzed to obtain fluorescent % and mean on all samples.

Immunofluorescence staining of frontal cortical biopsy samples
Biopsy samples were frozen in blocks of optimal cutting temperature (OCT, 25608-930, Tissue-Tek), sectioned at a thickness of 20 lm with a cryostat (Leica Microsystems, Wetzlar, Germany), collected on superfrost microscope slides (ThermoFisher Scientific, Waltham, USA) and stored at À70°C until analysis. First the iNPH biopsy samples were analyzed for the presence of Ab plaques. After air dry, the sections were rehydrated and washed twice with phosphate-buffer (PB) pH 7.4 and once with PBS for 5 min. For antigen retrieval, the sections were incubated for 45 min in preheated (92°C) 10 mM sodium citrate buffer, pH 6.0 and the buffer was let cool down at room temperature for 45 min. The slides were washed three times for 5 min with PBS containing 0.05% Tween-20 (PBST; Sigma-Aldrich, St. Louis, USA). Endogenous peroxidase was blocked using 0.3% H 2 O 2 in MeOH for 30 min at room temperature before three washes as previously. The sections were blocked by 1 h incubation in 10% normal horse serum (Vector Laboratories Ltd., Burlingame, USA). Next, samples were incubated at room temperature with rabbit anti-Ab, (dilution 1:800, 8243s, Cell Signaling Technology) in 5% normal horse serum in PBST. After three 5 min washes with PBST, the samples were incubated for 2 h with biotinylated goat anti-rabbit IgG antibody (dilution 1:200, BA-1000, Vector Laboratories) in 5% normal horse serum in PBST. After three 5 min washes with PBST, the slides were incubated in ABC reagent (PK-6101, Vector Elite Kit, Vector Laboratories) for 2 h at room temperature. After washes with PBST as above, the sections were incubated with Ni-DAB in 0.075% H 2 O 2 until the color developed. The reaction was stopped by washing in ddH 2 O twice for 7 min. The sections were dehydrated in ethanol gradient (50%, 70%, 95%, 100% EtOH) and 2 times with xylene for 1 min each and embedded using DePex (Serva) on coverslips.
For double and triple immunohistochemical staining the sections were air dried, washed, and boiled for antigen retrieval similarly to Ab staining. Next, the slides were washed three times with PBST, blocked for lipofuscin with 1:20 dilution of TrueBlackâ Lipofuscin Autofluorescence Quencher (Cat# 23007, Biotium, San Francisco, USA) in 70% ethanol for 1 min and then washed three times with PBST. The sections were blocked for 1 h in 5% BSA or 10% normal horse serum (Vector Laboratories Ltd., Burlingame, USA). The primary antibodies were incubated overnight at room temperature: goat anti-factor H (dilution 1:400; Cat# 341276, Calbiochem/Sigma-Aldrich, St. Louis, USA), rabbit anti-apolipoprotein E (dilution 1:400; Cat# ab183597; Abcam, Cambridge, UK), rabbit anti-C1q (dilution 1:300, Cat#A0136, Dako), mouse anti-Ab (dilution 1:1,000; clone WO-2; Cat# MABN10, Millipore), rabbit anti-Ab (dilution 1:800; clone D54D2; Cat# 8243s, Cell signaling technology) and rabbit anti-Iba1 (Cat#. 019-19741, Fujifilm Wako) in 3% BSA or 5% normal horse serum in PBST. For double and triple IHC stainings, antibodies for FH and/or apoE together with antibody against Ab were used. After washing in PBST, the sections were incubated with 1:500 dilution of Alexa Fluor 488 (chicken anti-Rabbit IgG, Invitrogen, Cat# A21441), 488 (donkey anti-goat 488 Invitrogen Cat#A11055), 555 (donkey anti-mouse IgG, Invitrogen, Cat#31570), 594 (donkey anti-goat IgG, Invitrogen, Cat# A11058) 594 (donkey anti-rabbit 594 Invitrogen Cat# A21207) or 680 (donkey anti-goat IgG, Invitrogen, A21084) secondary antibody (ThermoFisher Scientific, Waltham, USA) for 2 h at room temperature in 5% normal horse serum in PBST, washed again, and mounted in Vectashield with DAPI (Cat# H1200, Vector Laboratories Ltd., Burlingame, USA). Negative controls were included in parallel sessions following the same procedures without the incubation with primary antibodies. The confocal images were imaged under × 20, × 40 or × 64 magnification with Zeiss Axio Observer with Zeiss LSM 800 Airyscan confocal module (Carl Zeiss AG, Jena, Germany) and analyzed using the Zen software (all Carl Zeiss AG, Jena, Germany). For colocalization analysis and for measuring microglia Iba-1 and Ab intensities the DAPI channel was segmented using Otsu's method with any areas smaller than 50 pixels or holes removed using morphological transformations. Unique cell regions were defined based on each separate mask from the DAPI channel. A bounding box of 100 pixels was drawn around each mask and the whole region of interest correlation coefficient calculated between the channels or intensities for the microglia (Iba-1), Ab and FH, the summed background corrected pixel intensity was calculated.

Mass spectrometry
The frontal cortex biopsy samples were from apoE genotyped iNPH patients that were analyzed for the presence or absence of Ab plaques. These included three apoE44 Ab positive, three apoE43 Ab positive, two apoE43 Ab negative, four apoE33 Ab positive, three apoE33 Ab negative and two apoE23 Ab negative samples. All of the collected brain biopsy was taken for homogenization. Homogenization was done in 6.0 M urea/50 mM NH 4 HCO 3 (Sigma-Aldrich) by sonicating the samples in an ice bath with 15 min of continuous sonication followed by a 10 min break for a total of two cycles. Then samples were centrifuged at 14,000 g for 15 min at 4°C and supernatant was collected. For total protein concentration measurement urea concentration was diluted to 3 M with 50 mM NH 4 HCO 3 . Total protein concentration of the samples was measured with BCA protein assay kit (Pierce, Thermo Scientific) and 50 lg of total protein from each sample was taken for in-solution digestion. The proteins were reduced with 5 mM DL-dithiothreitol (Sigma Aldrich) alkylated with 15 mM iodoacetamide (Fluka), and trypsin-digested with 2 ll Sequencing Grade Modified Trypsin (Promega) overnight at 37°C. Desalting was done with C18 MicroSpin Columns (The Nest Group, cat. no. SEMSS18V). After the C18 purified peptides were dried, the samples were resuspended into 30 ll of buffer A (1% ACN, 0.1% FA in HPLC grade H 2 O; all from Merck). Samples were diluted 1:50 prior to loading to EvoTips (EvoSep, Denmark). The total volume of 20 ll was pipetted into EvoTips before analysis by EvoSepOne with EvoSep EV1109 analytical column using a 60 samples/day method coupled to timsTOF Pro (Bruker, Germany) operated with DDA PASEF-short gradient 0.5 s cycletime -method.
The resulting MS files were submitted to the FragPipe v16.0 software for protein identification and label-free quantification. The FragPipe v16.0 included MSFragger v3.3 and Philosopher v4.0.0. The searches were performed with reviewed Human proteome UP000005640. Decoy sequences and common contaminants were generated and added to the original database as part of the FragPipe workflow. Trypsin was selected as the cleavage specificity and methionine oxidation and N-terminal acetylation were set as variable modifications. Static residue modification was set for carbamidomethylation of cysteines. The allowed peptide length and mass ranges were 5-50 residues and 200-5,000 Da, respectively. Within FragPipe all peptide-spectrum matches (PSMs), peptides, and proteins were filtered to 1% PSM and 1% protein FDR. For MSFragger precursor tolerance was set to 50 ppm and fragment tolerance was set to 20 ppm, with mass calibration and parameter optimization enabled. Two missed cleavages were allowed, and two enzymatic termini were specified. Isotope error was set to 0/1/2. The minimum number of fragment peaks required to include a PSM in modeling was set to two, and the minimum number required to report the match was four. The top 150 most intense peaks and a minimum of 15 fragment peaks required to search a spectrum were used according to recommended settings. Label-free quantification was employed with default settings.

Crosslinking of proteins
For crosslinking, 2 lg of FH 5-7 protein was mixed with 3 lg of apoE2 N-terminal fragment (1-165) at 1:1 molar ratio in a final reaction volume of 20 ll in PBS and incubated for 15 min at 37°C, at 1,000 rpm shaking speed for the proteins to bind to each other. Heavy (deuterated) and light disuccinimidyl suberate (DSS-H12/ DSS-D12, Creative Molecules Inc., 001S) was added to final concentrations of 0.25 mM and the samples further incubated for 60 min at 37°C, at 1,000 rpm shaking speed. The crosslinking reaction was quenched with a final concentration of 50 mM of ammonium bicarbonate for 15 min at 37°C, 1,000 rpm.

Crosslinked sample preparation for mass spectrometry analysis
All samples for MS analysis were prepared by denaturing the proteins using 8 M urea -100 mM ammonium bicarbonate solution. The cysteine bonds were reduced with a final concentration of 5 mM Tris(2carboxyethyl) phosphine hydrochloride (TCEP, Sigma, 646547) for 60 min at 37°C, 800 rpm and subsequently alkylated using a final concentration 10 mM 2-iodoacetamide for 30 min at 22°C in the dark. For digestion, 1 lg of lysyl endopeptidase (LysC, Wako Chemicals, 125-05061) was added, and the samples incubated for 2 h at 37°C, 800 rpm. The samples were diluted with 100 mM ammonium bicarbonate to a final urea concentration of 1.5 M, and 1 lg of sequencing grade trypsin (Promega, V5111) was added for 20 h at 37°C, 800 rpm. The digested samples were acidified with 10% formic acid to a final pH of 3.0. Peptides were purified and desalted using C18 reverse phase column following the manufacturer's recommendations (The Nest Group, Inc.). Dried peptides were reconstituted in a solution containing 2% acetonitrile and 0.1% formic acid prior to MS analysis.

Liquid chromatography mass spectrometry for crosslink identification
A total of 500 ng of peptides were analyzed on an Orbitrap Eclipse mass spectrometer connected to an ultra-high-performance liquid chromatography Dionex Ultra300 system (Thermo Scientific). The peptides were loaded and concentrated on an Acclaim PepMap 100 C18 precolumn (75 lm × 2 cm) and then separated on an Acclaim PepMap RSLC column (75 lm × 25 cm, nanoViper, C18, 2 lm, 100 A; both columns Thermo Scientific), at a column temperature of 45°C and a maximum pressure of 900 bar. A linear gradient of 2-25% of 80% acetonitrile in aqueous 0.1% formic acid was run for 100 min followed by a linear gradient of 25-40% of 80% acetonitrile in aqueous 0.1% formic acid for 20 min. One full MS scan (resolution 120,000; mass range of 400-1,600 m/z) was followed by MS/MS scans (resolution 15,000) of the 20 most abundant ion signals. Precursors with an unknown charge state, a charge state of 1, 2, or above 8 were excluded. The precursor ions were isolated with 1.6 m/z isolation window and fragmented using higher-energy collisional-induced dissociation (HCD) at a normalized collision energy of 30. The dynamic exclusion was set to 45 s.

Crosslinking data analysis
All spectra from crosslinked samples were analyzed using pLink 2 (version 2.3.10; DOI 10.1038/s41467-019-11337-z). The target protein database contained the sequence for the human FH 5-7 and ApoE2 proteins. pLink2 was run using default settings for conventional HCD DSS-H12/D12 crosslinking, with trypsin as the protease and up to three missed cleavages allowed. Peptides with a mass range of 600-6,000 m/z were selected (peptide length of ca. 6-60 residues) and the precursor and fragment tolerance were set to 20 and 20 ppm, respectively. Crosslink identifications were filtered by requiring 10 ppm mass accuracy, false discover rate (FDR) < 5%, E-value < 0.01, and ≥ 5 observed spectra, including spectra with both DSS-H12 and DSS-D12. The unfiltered crosslinking data are presented in Dataset EV1. All crosslinking data have been deposited to the ProteomeXchange consortium via the MassIVE partner repository https://massive.ucsd.edu/ with the dataset identifier PXD039369.

Molecular docking and molecular dynamics simulations
The FH5-7/ApoE2 complex was modeled with the crosslink data as restraints with HADDOCK v2.4 web server (https://wenmr.science. uu.nl/haddock2.4/; van Zundert et al, 2016) with maximum DSS crosslink distance as 30 A (Merkley et al, 2014) between the crosslinked lysine residues. This resulted in five clusters, of which four representative structures of each cluster were submitted to atomistic molecular dynamics simulations in explicit solvent. These 20 structures were solvated, thoroughly energy minimized, equilibrated, and finally simulated using two complementary atomistic force fields, CHARMM36m  and Amber FF14SB (Maier et al, 2015), for 250 ns each using the GROMACS 2021 simulation engine (Pall et al, 2020). The recommended simulation parameters for these force fields were used. The stability of the interfaces was assessed by the mean RMSD of FH5-7 during the last 50 ns of simulation after first RMSD-fitting apoE2 the structure to its initial conformation. For the most stable structure, the key hydrogen bonds predicted by both force fields were analyzed. For details on the simulation setup and simulation parameters, see Fig EV2.

Statistical analysis
For MS1 intensity fold change calculations, samples were grouped based on APOE genotypes. After discarding proteins for which no valid values in at least 50% of one group were found, the MS1 intensities were log2 transformed, missing values were imputed from a normal distribution using the QRILC method, and fold changes and P-values calculated using the DEP R package (Zhang et al, 2018). To analyze expression of complement activation markers, the MS1 intensities of the markers were normalized against neutral apoE33 samples, and the combined intensity values were compared. Statistical significances were calculated from experiments that were performed using at least three biological replicates by SPSS statistics (SPSS version 24 IBM Statistics). First, Kolmogorov-Smirnov normality test was used to analyze, whether variables were normally distributed. For multiple comparisons and samples with unequal variances, two-tailed one-way ANOVA supplemented with Dunnett's post hoc test was used (SPSS version 24 IBM Statistics). Standard P-value threshold of < 0.05 was used to indicate statistical significance. For the differential expression comparison, DESeq2 was used with the threshold P-value set to 0.05 (Bioconductor). Pathway analysis was using Ingenuity Pathway Analysis software (IPA, Qiagen). Band intensities in WB and PAGE gels were analyzed using ImageJ software version 1.53c (National Institutes of Health, USA). MST statistics was done using MO. Affinity Analysis Software (NanoTemper). Flow cytometric analysis was done using FlowJo v10.1r5 software (FlowJo). The single molecules TIRF microscopy data were analyzed using bespoke MATLAB software to track and quantify the intensity of foci as a function of time where statistics of multiple images was calculated using twotailed Student's t-test.
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