Carboxyl-terminal-truncated forms of apoE accumulate in AD brains and NFTs. (a and b) Immunostaining of AD brain sections with anti-amino-terminal apoE (a) or anti-carboxyl-terminal apoE (b). (c and d) Double immunofluorescence staining of AD brain sections with anti-p-tau (red) and anti-amino-terminal apoE (green) (c) or anti-p-tau (red) and anti-carboxyl-terminal apoE (green) (d). (e and f) Neuro-2a cells expressing apoE3 or apoE4 were incubated with or without synthetic Aβ_1–42 (10 μM) for 24 h at 37°C. After incubation, cell lysates were immunoprecipitated with a monospecific polyclonal anti-apoE followed by Western blotting with polyclonal anti-apoE (e) or monoclonal anti-apoE (6C5) (f) that recognizes the first 15 amino acids of the amino terminus of apoE (45). (g–k) Brain tissues from two nondemented normal individuals (N1 and N2) and three AD patients (AD1, AD2, and AD3) were homogenized. Both the supernatants (g and h) and the solubilized pellets (i–k) were subjected to SDS/PAGE and analyzed by Western blotting with polyclonal antibodies against full-length apoE (g and i), the carboxyl terminus (amino acids 272–299) of apoE (h and j), or a mAb against p-tau (AT8) (k). E Std, apoE standard (200 ng protein). (Original magnifications: a and b, ×200; c and d, ×600.)

Carboxyl-terminal-truncated apoE induces intracellular NFT-like inclusions in the cytosol of Neuro-2a cells. (a) Neuro-2a cells transiently transfected with apoE4 cDNA were double-immunostained with anti-apoE (green) and anti-p-tau (red) antibodies. This is representative of the cells containing apoE and p-tau immunoreactive intracellular inclusions (yellow) (more than 1,500 cells examined). (b) ApoE3- and apoE4-transfected Neuro-2a cells were incubated with or without 10 μM synthetic Aβ_1–42 peptide at 37°C for 36 h. After incubation, the cells were double-immunostained with anti-apoE and anti-p-tau, and the percentage of transfected cells containing apoE and p-tau immunoreactive intracellular inclusions was calculated. Each column represents mean ± SD of four experiments (P < 0.001, apoE4 versus apoE3 or apoE4 + Aβ_1–42 versus apoE3 + Aβ_1–42; >400 transfected cells counted per experiment). (c) Cell lysates of apoE4-transfected Neuro-2a cells treated with Aβ_1–42 (10 μM, 36 h) were immunoprecipitated with a monoclonal anti-p-tau (AT8 or AT270). Control lysates were not immunoprecipitated with anti-p-tau. Anti-apoE Western blotting revealed a band with a lower molecular mass than full-length apoE (≈29–30 kDa versus 34 kDa). (d) Neuro-2a cells were incubated for 30 h at 37°C with recombinant apoE4(Δ272–299) (30 μg/ml) complexed with rabbit β-very low density lipoproteins (20 μg of protein/ml). After incubation, the cells were fixed and immunostained for apoE. (e–h) Neuro-2a cells transiently transfected with apoE4(Δ272–299) (e–g) or apoE3(Δ272–299) (h) cDNA were double-immunostained with anti-apoE (e) and anti-p-tau (f) antibodies or immunostained with anti-apoE alone (h). (g) Merged image of e and f. (i–l) Neuro-2a cells were transiently transfected with DNA constructs encoding GFP (i), GFP-apoE3(Δ272–299) (j), or GFP-apoE4(Δ272–299) (k). All three constructs lacked the sequence encoding the signal peptide, resulting in direct expression of GFP-apoE in the cytosol. After transfection, the percentage of transfected cells containing intracellular filamentous inclusions was calculated (l) (mean ± SD of four experiments; >300 transfected cells counted per experiment. P < 0.001, GFP-apoE4(Δ272–299) versus GFP-apoE3(Δ272–299). (Original magnification: a and d–k, ×600.)

Electron micrograph of the intracellular inclusions induced by GFP-apoE4(Δ272–299) in Neuro-2a cells. Neuro-2a cells were transiently transfected with GFP (a) or GFP-apoE4(Δ272–299) (b and c). GFP-positive cells were sorted with a fluorescence-activated cell sorter, pelleted by centrifugation, and examined with a JEOL CCX-100II electron microscope. (Original magnifications: a and b, ×25,000; c, ×60,000.)

Intracellular inclusions induced by GFP-apoE4(Δ272–299) in Neuro-2a cells contain p-tau and p-NF-H. (a–f) Neuro-2a cells transiently transfected with GFP-apoE4(Δ272–299) were immunostained for p-tau (AT270; similar results were obtained with AT8) or p-NF-H (e, SM31; similar results were obtained with RT97) and imaged by confocal microscopy. (c) Merged image of a and b. (f) Merged image of d and e. (g–i) Detection of a complex containing p-tau (g), p-NF-H (h), and truncated apoE (i) in Neuro-2a cells transiently transfected with apoE4(Δ272–299) after immunoprecipitation with anti-apoE (g and h) or anti-p-tau (AT8) (i) and Western blotting. (Original magnification: a–f, ×600.)

The effects of deletions in the carboxyl- and amino-terminal domains of apoE on the formation of NFT-like structures in Neuro-2a cells. DNA constructs encoding fusion proteins of GFP and various carboxyl- or amino-terminal-truncated forms of apoE4 were prepared and transiently transfected into Neuro-2a cells. All constructs lacked the signal peptide sequence. (a) Model of apoE (modified from ref. 46) illustrating the structural regions where deletions were made and the polymorphic site (residue 112) that distinguishes apoE3 from apoE4. (b) Results defining amino acids 245–260 of apoE as critical for inclusion formation. (c) Deletion of helix 3 (amino acids 86–126) significantly reduced the ability of apoE4(Δ272–299) to form the intracellular inclusions (P < 0.001). The percentages of cells containing NFT-like structures were determined (mean ± SD of four experiments; >300 transfected cells counted per experiment).