The Molecular Basis for Apolipoprotein E4 as the Major Risk Factor for Late-Onset Alzheimer's Disease

Abstract

Apolipoprotein E4 (ApoE4) is one of three (E2, E3 and E4) human isoforms of an α-helical, 299-amino-acid protein. Homozygosity for the ε4 allele is the major genetic risk factor for developing late-onset Alzheimer's disease (AD). ApoE2, ApoE3 and ApoE4 differ at amino acid positions 112 and 158, and these sequence variations may confer conformational differences that underlie their participation in the risk of developing AD. Here, we compared the shape, oligomerization state, conformation and stability of ApoE isoforms using a range of complementary biophysical methods including small-angle x-ray scattering, analytical ultracentrifugation, circular dichroism, x-ray fiber diffraction and transmission electron microscopy We provide an in-depth and definitive study demonstrating that all three proteins are similar in stability and conformation. However, we show that ApoE4 has a propensity to polymerize to form wavy filaments, which do not share the characteristics of cross-β amyloid fibrils. Moreover, we provide evidence for the inhibition of ApoE4 fibril formation by ApoE3. This study shows that recombinant ApoE isoforms show no significant differences at the structural or conformational level. However, self-assembly of the ApoE4 isoform may play a role in pathogenesis, and these results open opportunities for uncovering new triggers for AD onset.

Keywords: Alzheimer's disease; alpha-helix; analytical ultracentrifugation; apolipoprotein E; small-angle x-ray scattering.

Graphical abstract

Fig. 1

Characterization of recombinant ApoE2, E3 and E4. (a) The SEC elution pattern of ApoE isoforms on the Superdex 200 column as a function of absorbance at 280 nm versus the elution volume. All ApoE isoforms have a minor elution peak at 131 mL and a major at 151 mL suggesting the presence of different oligomeric species. A hydrodynamic radius of 5.8 nm, as well as a diffusion coefficient of 2.47 × 10⁻⁷ cm s⁻¹ is calculated by calibration of the Superdex 200 column (inlet) using commercially available protein standards (1, ferritin; 2, aldolase; 3, conalbumin; 4, ovalbumin). A frictional ratio f/f₀ = R_S/R_min of 1.71 is calculated, which suggest moderate elongation of ApoE . (b) SEC-MALS plotted with the differential refractive index (RIU; line) are shown as a function of elution time for ApoE2, ApoE3 and ApoE4. Inset shows the calculated molecular weight (MW) across the peak. All ApoE isoforms have identical elution volumes, and a MW of 145, 139 and 151 kDa is calculated at elution peak for ApoE2, ApoE3 and ApoE4, respectively. (c and d) AUC showing continuous c(S) size distributions in size exclusion buffer [20 mM Hepes, 300 mM NaCl and 10 % (v/v) glycerol pH 8.0; (c)] and in 20 mM PB (pH 7.4; (d). No difference between ApoE isoforms is observed in either buffer condition. A major species with a sedimentation coefficient S at 3 and 5 is observed in size exclusion and PB buffer, respectively. The difference in sedimentation coefficient between buffers is due to the presence of glycerol in the SEC buffer.

Fig. 2

SAXS. X-ray scattering curves and the dimensionless Kratky plot (ScÅtter) for ApoE2 (a), ApoE3 (b) and ApoE4 (c) are shown, as well as their corresponding pair distance distribution function (d) P(r). (a–c) All ApoE isoforms have identical scattering profiles and adopt an extended conformation in solution with some intrinsic level of flexibility as assessed by the dimensionless Kratky plot. (d) This extended conformation is seen in the P(r) distribution, respectively, and a maximal dimension D_max of approximately 19.5 nm (195 Å) is determined for each isoform.

Fig. 3

Conformation and stability of ApoE isoforms. (a) Far UV CD spectra of ApoE isoforms (25 μM in 20 mM PB, pH 7.4, 21 °C) showing comparable α-helical content. Secondary structure analysis was conducted with CONTIN/LL , at DichroWeb , , using the reference data set 6 , and results can be found in Table 3. (b) Intrinsic tryptophan fluorescence (excitation at 295 nm) indicates that all three isoforms have a similar tertiary structure in PB. (c) Changes in helical content were followed at 222 nm with increasing temperature. A Boltzmann sigmoidal equation was fitted to the data, showing the order of stability E2 >> E3 >= E4 (i). The fraction unfolded of each protein calculated from the fitted curves was also plotted against temperature (ii). Tm (°C) for each isoform, the temperature at which there was 50% change in α-helical content, estimated from the fit denaturation curves can be found in Table 4a. Comparable α-helical content at 37°C were also reported in Table 4a, and fraction unfolded at 37° C is shown in Table 4b. (d) GuHCl chemical denaturation of recombinant ApoE isoforms plotted showing the wavelength of maximum fluorescence λ_max at an excitation of 292 nm. Experimental data points (closed circles) are shown. (i) A three-state unfolding model was fitted to the data; apparent midpoint GuHCl concentrations can be found in Table 4b. (ii) Calculated fraction unfolded for each isoform from the fitted three-state unfolding model is shown. ApoE2 (blue connective line), ApoE3 (green connective line), ApoE4 (red connective line).

Fig. 4

Self-assembly of ApoE4 but not ApoE2 or ApoE3. (a) Native gel shows the formation of higher oligomeric species for ApoE4 (> 1048 kDa) but not E2 and E3 after incubation at 37°C for 24 h. (b) Kinetics of ApoE self-assembly (ApoE2, blue; ApoE3, green; ApoE4, red) was monitored by recording changes in ThT fluorescence intensity at 483 nm over 3 days. A comparison between the three isoforms was established by looking at adjusted ThT fluorescence. Changes in ThT fluorescence varied with each isoform, with a faster increase observed for ApoE4. After 3 days, ApoE3 and ApoE4, but not ApoE2, show changes in ThT fluorescence (top panel). The bottom panel is a close-up on the first 24 h of assembly. The length of the lag phase was different for each isoform, with ApoE2 > ApoE3 >> ApoE4. Envelopes correspond to the standard error of the mean (SEM). (c) TEM of negatively stained ApoE isoforms after incubation for 24 h (left side) and 3 days (right side) at 37 °C shows no fibril formation of ApoE2 and ApoE3. ApoE4 self-assembles to form long, curved fibrils. ApoE3 formed round oligomeric species after a 3-day incubation. The scale bars represent 500 nm.

Fig. 5

Characterization of ApoE4 fibrils. (a) Transmission electron micrographs of negatively stained ApoE4 (25 μM in 20 mM PB, pH 7.4) monitored over 24 h at 37 °C. The scale bar represents 200 nm. ApoE4 self-assembly was characterized by measuring changes in length (b) and width (c) of the fibrils. Changes in width were non-significant; however, with increasing incubation times, ApoE4 fibrils become significantly longer (average length of 363 nm; one-way ANOVA: ****p < 0.0001, F = 180.9). (d) CD spectra show retention of α-helical secondary structure after assembly (whole fraction). Fibrils in the pellet were separated from the supernatant to confirm that their secondary structure is not masked by protein in the supernatant. Fibrils in the pellet showed an α-helical conformation. (e) X-ray fiber diffraction pattern obtained from partially aligned ApoE4 fibrils after 24-h incubation at 37 °C showing positions of diffraction signals on the meridian (vertical) and equatorial (horizontal) axes.

Fig. 6

ApoE3 inhibits ApoE4 fibril formation. (a) Adjusted ThT fluorescence at 483 nm was monitored over the course of 24 h, 37 °C for ApoE3 alone (25 μM), ApoE4 alone (25 μM) and ApoE3 plus ApoE4 (both 12.5 μM for a total ApoE concentration of 25 μM) in PB (pH 7.4). (b) Transmission electron micrographs showed a heterogeneous population, with the presence of small round and small amorphous species, but no mature fibrils when E4 is incubated with E3 in a 1:1 ratio. The scale bar represents 500 nm.