The role of complement FH and apoE interactions in the regulation of Aβ1-42 and C1q-mediated complement activation

The APOE4 gene variant of apolipoprotein E (apoE) has been identified as a predisposing factor for late-onset Alzheimer’s disease (AD). ApoE is known to interact with one of the classically characterized hallmarks of AD, the amyloid-beta (Aβ) protein. Aβ activates the classical complement pathway by binding to C1q that may sustain inflammation, hamper Aβ clearance and therefore promote accumulation of Aβ deposits in the brain and affect the integrity of the blood-brain barrier. Depending on context, binding of apoE to C1q can either activate or inhibit complement activation. ApoE can also interact with the complement regulator factor H (FH) which colocalizes with C1q on Aβ plaques. The interactions between these proteins and their role in the AD pathogenesis has not been fully explored. This thesis aimed at unraveling the complex formation between Aβ1-42, apoE, FH, and C1q, and their role in complement activation to provide novel insights on the mechanisms contributing to AD pathophysiology. By conducting enzyme-linked immunosorbent assays and Western Blotting of native PAGE, I was able to show that FH bound to apoE and forms a complex with Aβ1-42/apoE in an isoform- specific manner (apoE2 > apoE3 > apoE4) whereas C1q bound all Aβ1-42/apoE complexes with same affinity. FH did not bind Aβ1-42 alone, thus the results indicated that binding of FH in Aβ1-42/apoE complex took place via apoE2 and apoE3. In the absence of FH and C1q, immobilized Aβ1-42/apoE4 complexes led to higher serum complement activation levels when compared to Aβ1-42/apoE2 and Aβ1-42/apoE3 complexes or Aβ1-42 alone. C1q activated serum complement especially in the presence of Aβ1-42/apoE3 and Aβ1-42/apoE4. Further functional analysis with flow cytometry revealed that in fluid phase the presence of FH could restrict Aβ1-42-induced complement activation, the subsequent formation of C5a and activation of C5a receptor (C5aR) expressed on Human embryonic kidney (HEK) 293 cells. The presence of all apoE isoforms on Aβ1-42/C1q complexes led to reduced C5aR activation. Surprisingly, however, the reduction was the largest with apoE4, and the presence of C1q on Aβ1-42 alone could also decrease C5aR activation. The results of this thesis elucidated the effects of Aβ1-42, apoE, C1q and FH in complement activation and proposed a mechanism by which FH regulates complement activation and cell-mediated inflammation through isoform-specific binding to apoE associated with Aβ1-42. Further work is required to analyze the functional effects of these complexes, whether Aβ1-42, apoE, FH, and C1q can all bind simultaneously to form a large complex and whether this complex affects C5aR activation. This study provides important insights on how APOE4 may predispose to the neurotoxicity and neuroinflammatory changes in AD pathophysiology early in the disease process. Yet, the findings are only preliminary and therefore further studies are required to validate these results and to demonstrate whether promoting FH binding to apoE/Aβ complex could provide a novel target for AD therapy