Altering APP Proteolysis: Increasing sAPPalpha Production by Targeting Dimerization of the APP Ectodomain

One of the events associated with Alzheimer's disease is the dysregulation of α- versus β-cleavage of the amyloid precursor protein (APP). The product of α-cleavage (sAPPα) has neuroprotective properties, while Aβ1-42 peptide, a product of β-cleavage, is neurotoxic. Dimerization of APP has been shown to influence the relative rate of α- and β- cleavage of APP. Thus finding compounds that interfere with dimerization of the APP ectodomain and increase the α-cleavage of APP could lead to the development of new therapies for Alzheimer's disease. Examining the intrinsic fluorescence of a fragment of the ectodomain of APP, which dimerizes through the E2 and Aβ-cognate domains, revealed significant changes in the fluorescence of the fragment upon binding of Aβ oligomers—which bind to dimers of the ectodomain— and Aβ fragments—which destabilize dimers of the ectodomain. This technique was extended to show that RERMS-containing peptides (APP695 328–332), disulfiram, and sulfiram also inhibit dimerization of the ectodomain fragment. This activity was confirmed with small angle x-ray scattering. Analysis of the activity of disulfiram and sulfiram in an AlphaLISA assay indicated that both compounds significantly enhance the production of sAPPα by 7W-CHO and B103 neuroblastoma cells. These observations demonstrate that there is a class of compounds that modulates the conformation of the APP ectodomain and influences the ratio of α- to β-cleavage of APP. These compounds provide a rationale for the development of a new class of therapeutics for Alzheimer's disease

Direct Transcriptional Effects of Apolipoprotein E

A major unanswered question in biology and medicine is the mechanism by which the product of the apolipoprotein E ε4 allele, the lipid-binding protein apolipoprotein E4 (ApoE4), plays a pivotal role in processes as disparate as Alzheimer's disease (AD; in which it is the single most important genetic risk factor), atherosclerotic cardiovascular disease, Lewy body dementia, hominid evolution, and inflammation. Using a combination of neural cell lines, skin fibroblasts from AD patients, and ApoE targeted replacement mouse brains, we show in the present report that ApoE4 undergoes nuclear translocation, binds double-stranded DNA with high affinity (low nanomolar), and functions as a transcription factor. Using chromatin immunoprecipitation and high-throughput DNA sequencing, our results indicate that the ApoE4 DNA binding sites include ∼1700 gene promoter regions. The genes associated with these promoters provide new insight into the mechanism by which AD risk is conferred by ApoE4, because they include genes associated with trophic support, programmed cell death, microtubule disassembly, synaptic function, aging, and insulin resistance, all processes that have been implicated in AD pathogenesis. SIGNIFICANCE STATEMENT This study shows for the first time that apolipoprotein E4 binds DNA with high affinity and that its binding sites include 1700 promoter regions that include genes associated with neurotrophins, programmed cell death, synaptic function, sirtuins and aging, and insulin resistance, all processes that have been implicated in Alzheimer's disease pathogenesis

Binding of Aβ fragments to eAPP_230–624 can be measured using intrinsic fluorescence.

<p>A) Emission spectra from the titration of 0.64 µM eAPP_230–624 with 7-kDa Aβ 1-40 from 0–13 µM (1∶0–1∶20 molar ratio) using the Shimadzu RF-530PC. The black line indicates the starting concentration and the gold line indicates the final concentration. The arrow indicates the direction of shift of the fluorescence peak. B) Emission spectra from the titration of 0.64 µM eAPP_230–624 with Aβ 10–20 from 0–46 µM (1∶0–1∶70 molar ratio) using the Shimadzu RF-530PC. The black line indicates the starting concentration and the blue line indicates the final concentration. The arrow indicates the direction of shift of the fluorescence peak. C) Summary of the results of titrating 0.64 µM eAPP_230–624 with various fragments of Aβ that inhibit eAPP dimerization: Aβ1-40 (black circles), Aβ1-28 (black diamonds), Aβ12-28 (black triangles), Aβ 10–20 (black squares), Aβ12-24 (yellow squares), Aβ5-14 (yellow diamonds) and Aβ16-20 (yellow circles) from 0–60 µM (1∶0–1∶85 molar ratio depending on the fragment). The excitation wavelength is 295 nm and the emission wavelength is 345 nm. To exclude the possibility that Aβ1-28 or Aβ10-20 formed oligomers, samples of these peptides were analyzed using size-exclusion chromatography as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040027#pone-0040027-g003" target="_blank">Fig. 3A</a>. Both of these peptides eluted 0.5 mls later than the 7-kDa peak, indicating that they are smaller dimers or monomers.</p

Disulfiram inhibits dimerization of MBP-eAPP_230–624.

<p>A) Small angle x-ray scattering analysis of MBP-eAPP_230–624 incubated with 50 µM disulfiram or 50 µM sulfiram at a 1∶12 molar ratio in PBS plus 2% DMSO. Incubation with either disulfiram or sulfiram produces significant deviations in the scattering curves indicating a significant shift in size and shape. The Guinier plot of MBP-eAPP_230–624 (black), sulfiram-treated (blue), and disulfiram-treated (red) is shown in the inset. The y-axis of the Guinier plot is normalized so that one unit is proportional to 90-kDa (the molecular mass of MBP-eAPP_230–624). The molecular mass of the complex can be obtained from the extrapolated y-intercept of the best-fit line. The downward shift of the line indicates that both sulfiram and disulfiram shift the monomer-dimer equilibrium in favor of the monomer. The curvature of the Guinier plot for disulfiram is evidence that the sample is a mixture of monomer and dimer. B) Cleavage of MBP-eAPP_230–624 with ADAM10 as measured by AlphaLisa.</p

Disulfiram and Sulfiram bind to eAPP_230–624.

<p>A) Difference absorption spectra of eAPP_230–624 in the presence of sulfiram (blue) and disulfiram (dotted red) at a 1∶6 molar ratio. For each difference curve, the absorption spectra of PBS +2% DMSO, PBS plus 2% DMSO and 50 µM sulfiram or PBS plus 2% DMSO and 50 µM disulfiram were subtracted from the absorption spectra of 8.3 µM eAPP_230–624 in the same buffer. B) Emission spectra from the titration of 0.64 µM eAPP_230–624 with disulfiram from 0–130 µM (1∶0–1∶202 molar ratio) using the Shimadzu RF-530PC. The black line indicates the starting concentration and the red line indicates the final concentration. The arrow indicates the direction of shift of the fluorescence peak. C) Emission spectra from the titration of 0.64 µM eAPP_230–624 with sulfiram from 0–320 µM (1∶0–1∶500 molar ratio) using the Shimadzu RF-530PC. The black line indicates the starting concentration and the blue line indicates the final concentration. The arrow indicates the direction of shift of the fluorescence peak. E) Stern-Volmer plot for the intermolecular quenching of 0.64 µM eAPP_230–624 with either disulfiram (346 nm –red circles and 375 nm red diamonds) or sulfiram (346 nm–blue circles and 375 nm-blue diamonds).</p

Disulfiram and Sulfiram increase sAPPα production.

<p>A) Effect of treatment with disulfiram and sulfiram on sAPPα and Aβ production in 7W-CHO cells. 7W-CHO cells were seeded at 50,000 cells per well in a 96-well plate and allowed to grow for one day. The cell culture medium was then changed and the cells treated with 1 µM disulfiram, 1 µM sulfiram, or 0.01% DMSO (control). After 24 hours, the amount of sAPPα and sAPPβ produced in the medium was quantified using the AL231C and AL232C AlphaLISA kit from Perkin Elmer, modified using a custom biotinylated human APP antibody. B) Effect on the ratio of α to β cleavage in 7W-CHO cells, as calculated by dividing the amount of sAPPα to sAPPβ produced by the 50,000 7W-CHO cells during one day under the effect of either disulfiram, sulfiram or a DMSO control. C) Effect on the ratio of α to β cleavage in B103 cells. B103 neuroblastoma cells were seeded at 100,000 cells per well in a 96-well plate and allowed to grow for one day. The cell culture medium was then changed and the cells treated with 1 µM disulfiram, 1 µM sulfiram, or 0.01% DMSO (Control). After 24 hours, the amount of sAPPα and sAPPβ produced in the medium was quantified using the same modified AL231C and AL232C AlphaLISA kit from Perkin Elmer. The ratio of α to β cleavage was calculated by dividing the amount of sAPPα to sAPPβ produced by the 100,000 B103 cells during one day under the effect of the disulfiram, sulfiram or DMSO control.</p

SAXS analysis of eAPP fragments and their complexes.

<p>The maximum estimated error in the radius of gyration (R _g) is ±2 Å. The maximum estimated error in maximum dimension (D_max) is ±10 Å. Both values were calculated with the program GNOM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040027#pone.0040027-Svergun1" target="_blank">[31]</a>. The relative mass was calculated as the ratio of the apparent mass of the protein (MW_calc) to the expected mass derived from the protein sequence (MW_seq). The molecular weight derived from the protein sequence is 52-kDa for TRX-eAPP_290–624, 45-kDa for eAPP_230–624, and 90-kDa for MBP-eAPP_230–624. For globular, non-interacting proteins, the apparent mass can be estimated by comparing the extrapolated scattering of the sample at zero scattering angle (I(0)) to that of the reference protein albumin and the eAPP fragment dimer</p><p></p>where subscripts <i>un</i> and <i>ref</i> refer to the sample and the reference protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040027#pone.0040027-Feigin1" target="_blank">[56]</a>. The error is the calculated mass is primarily due to the uncertainty in the estimation of the concentrations. The maximum error was determined experimentally by replicates of within a much larger set of experiments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040027#pone.0040027-Libeu1" target="_blank">[18]</a> to be ±0.2, where the maximum error determines the range of values for which p<0.05. Monomeric proteins with 20–30% of their residues in random coil conformation, as expected for monomeric APP, have relative mass estimates around 1.3⁵¹.<p></p

1,8-ANS binds to eAPP_230–624.

<p>A) Difference absorption spectra of eAPP_230–624 in the presence of 1,8-ANS (purple) at a 1∶10 molar ratio. For each difference curve, the absorption spectra of PBS, PBS plus 1,8-ANS was subtracted from the absorption spectra of 3 µM eAPP_230–624 in the same buffer. The appearance of significant changes in the peak absorption near 280 is consistent with perturbations in the environment of some of the aromatic residues in eAPP_230–624 caused by the binding of 1,8-ANS. The broad absorption peak centered on 380 nm is characteristic of 1,8-ANS bound to protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040027#pone.0040027-Weber1" target="_blank">[55]</a>. B) Comparison of the excitation peak for the intrinsic fluorescence at 375 nm for 3 µM eAPP_230–624 alone (solid black) and in the presence of 1,8-ANS at molar ratio of 1∶20 protein to 1,8-ANS at 375 nm (dotted purple) and 500 nm (purple). This comparison shows that the presence of 1,8-ANS at a 1∶20 molar ratio is sufficient to redirect the absorbed photons in the region dominated by tryptophan absorption (285–300 nm) into emission peak of 1,8-ANS at 500 nm consistent with FRET between the tryptophans and bound 1,8-ANS molecules. C) Emission spectra of the titration of 0.64 µM eAPP_230–624 with 1,8-ANS from 0–120 µM (1∶0–1∶180 molar ratio) using the Shimadzu RF-530PC. The black line indicates the starting concentration and the purple line indicates the final concentration. D) Stern-Volmer plot for intermolecular quenching of the intrinsic fluorescence of eAPP_230–624 by 1,8-ANS at 24°C derived from titration data from the Shimadzu RF-530PC. E) Emission spectrum of 4 µM eAPP_230–624 in PBS with varying molar ratios of 1,8-ANS from 1∶0 (black) to 1∶55 (purple) obtained using the Spectramax XPS. F) Stern-Volmer plot for intermolecular quenching of the intrinsic fluorescence of eAPP_230–624 by 1,8-ANS at 24°C (purple) and 42°C (red) as measured on the Spectramax XPS. The protein concentration was 2 µM for E–F.</p

Interaction of RERMS peptides with eAPP.

<p>A) Emission spectra from the titration of 0.64 µM eAPP_230–624 with RERMS peptides from 0–270 µM (1∶0–1∶420 molar ratio) using the Shimadzu RF-530PC. The black line indicates the starting concentration and the red line indicates the final concentration. The arrow indicates the direction of shift of the fluorescence peak. B) Stern-Volmer plot for the intermolecular quenching of 0.64 µM eAPP_230–624 by the RERMS peptide for the emission wavelengths: 345 nm (purple diamonds) and 375 nm (black circles) C) Model of APP319-335 peptide bound to the E2 domain of APP derived from a dimer of an E2 domain fragment (residues 299 to 490) observed in its crystal structure protein data base entry 1RW6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040027#pone.0040027-Wang1" target="_blank">[25]</a>. The major interactions between the peptide and the protein are two hydrogen bonds involving Arg 328 and both polar and hydrophobic interactions between Met 335 and the hydrophobic pocket. The rim of the hydrophobic pocket into which Met 335 binds is 9 Å from the C-terminus of the E2 domain fragment. D) Superposition of the peptide model (pink) with the SAXS model. The green balls show the predicted path in the SAXS model of the chain that forms a bridge from the terminus of the E2 domain to the dimer interface containing the C-terminus of eAPP_230–624. The overlap of the surfaces shows the overlap between the peptide docked into its binding site and the predicted path of the eAPP_230–624 residues indicating that the two models are incompatible. This overlap suggests a mechanism by which binding of an RERMS peptide in the binding site predicted by the crystal structure could influence the stability of the dimer of eAPP_230–624.</p