Apparent reasons for “failure” in some previous anti-amyloid clinical trials.

Apparent reasons for “failure” in some previous anti-amyloid clinical trials.</p

Aβ can trigger AD.

(A) Proteolytic processing of APP by β-secretase and γ-secretase leads to the generation of Aβ protein. Red asterisks: mutations that cause familial AD; green asterisk: a protective mutation. Insert: typical amyloid plaques and neurofibrillary tangles of AD pathology. (B) One way to depict the amyloid cascade. Individual steps in the cascade may evoke distinct microglial responses. Aβ, amyloid β-protein; AD, Alzheimer disease; APP, amyloid precursor protein.</p

Relationship of plaque lowering and less cognitive decline across anti-Aβ antibody trials.

Graphical relationship between amyloid-lowering effect (abscissa) of various anti-Aβ antibodies (“compounds”) in individual completed therapeutic trials and their effects on clinical outcome on the CDR-SB test (ordinate). The trend line moving toward the lower left corner across these trials signifies the relationship. (Obtained from reference [74]). Aβ, amyloid β-protein; CDR-SB, Clinical Dementia Rating-Sum of Boxes; PET, positron emission tomography.</p

Pathology of early-onset and late-onset AD.

Similar Aβ protein and tau pathology in a tissue sample from a patient with “sporadic” late-onset AD and an individual with a mutation in PS1, encoding presenilin-1, which causes early-onset familial AD. Shown is the gyrus parahippocampalis immunostained with the antibodies 4G8, which labels Aβ, and AT8, which labels ptau. Figure kindly provided by Dr. Thomas Arzberger (Center for Neuropathology and Prion Research, Ludwig-Maximilians-Universität, Munich). Aβ, amyloid β-protein; AD, Alzheimer disease; ptau, phospho-tau.</p

Lysine modification increases αSyn immunoreactivity by strengthening attachment to blot membranes.

<p><b>A.</b> HEL cells were crosslinked <i>in vivo</i> with the cleavable crosslinker DSP (1 mM) in comparison to DMSO-only treatment (-). Cytosols were prepared (post-20,000 <i>g</i>) and boiled in sample buffer with 5% (v/v) β-mercaptoethanol (βME), which cleaves the disulfide bond of DSP (DSP_ßME: reduced DSP). Samples (30 µg) were blotted with αSyn antibody 15G7. Equal protein loading was visualized by blotting for DJ-1 and by Ponceau (Ponc.) staining. <b>B.</b> Primary cortical rat neurons (13 days <i>in vitro</i>) were treated with 1 mM DSP <i>in vitro</i> (cytosolic lysates) in comparison to DMSO-only (-) treatment. Cytosols (post 100,000 <i>g</i>) were boiled in sample buffer with 5% βME and blotted with αSyn antibody 2F12, as well as antibodies to βSyn, γSyn and β-tubulin (β-tub.). Ponceau (Ponc.) stain of the blot membrane is shown on the left. <b>C.</b> Human (H.s.; 15 µg) and mouse (M.m., 30 µg) brain homogenates (PBS soluble fraction; post 100,000 <i>g</i>) were treated <i>in vitro</i> with DSP or DMSO-only (-), followed by boiling in sample buffer containing 5% βME. Blots were developed with αSyn antibody Syn1 as well as antibodies to β-tubulin, β-actin, GAPDH and DJ-1. <b>D.</b> Purified recombinant αSyn and purified hen-egg lysozyme (lyz) were crosslinked at a concentration of 500 ng/µL with 2 mM DSP (DSP_ßME) in comparison to DMSO only (-), followed by boiling in sample buffer with 5% βME. After separation by SDS-PAGE, samples were transferred to PVDF membranes which were dried immediately (left panel: ‘no wash’) or after washing overnight in PBST (‘wash’). Dried membranes were briefly stained with Coomassie Blue. 14, 17 and 28 kDa molecular weight markers are visible in the outer left lane.</p

<i>In vitro</i> incubation of lysates with 2 mM DSP followed by βME reduction allows optimal αSyn detection.

<p><b>A.</b> HEL intact cells (<i>in vivo</i>) or lysates (<i>in vitro</i>) were incubated with DMSO only (-) as well as gradients of 0.5, 2.0 and 8.0 mM DSP and DTBP, respectively. Cytosols (post-100,000 <i>g</i>) were boiled in sample buffer/5% βME and blotted with αSyn antibody 2F12. Identical exposures of the same blot are shown; film was cut at dotted line. <b>B.</b> HEL cell cytosols (post-100,000 <i>g</i>) at three different protein concentrations (2.4, 3.3., 4.5 µg/µL) were incubated with DMSO only (-) as well as a gradient of 0.5, 2.0 and 8.0 mM DSP and DSG. Samples were normalized to 2.4 µg after quenching, then boiled in sample buffer plus 5% βME and blotted with αSyn antibodies Syn1 and 15G7 as well as an antibody to GAPDH. <b>C.</b> HEL cell cytosols (post-100,000 <i>g</i>) were treated with DMSO-only (-) or DSP, quenched, boiled in sample buffer plus 5% βME and analyzed by blotting for αSyn (mAb Syn1 and pAb C20), Calmodulin, DJ-1, Ran, 14-3-3 and β-actin.</p

DSP/βME treatment facilitates easy detection of αSyn from cultured cells.

<p><b>A.</b> Cytosols (post-100,000 <i>g</i>) of 3D5 αSyn tet-off cells in induced (3D5_I) or repressed state (3D5_R) as well as lysates of the parental neuroblastoma cell line M17D were crosslinked with 2 mM DSP, followed by boiling in sample buffer with 5% βME. Western blot analysis was performed for αSyn (C20), DJ-1 and β-actin. (Note that the DJ-1 blot shows residual C20 signal from a previous exposure.) <b>B.</b> Cytosols (post-100,000 <i>g</i>) of neuroblastoma cell lines M17D and SH-SY5Y as well as the PBS-soluble fraction of human brain homogenates were incubated in 2 mM DSP, followed by boiling in sample buffer with 5% βME and blotting for αSyn (2F12) as well as β-tubulin. A long and a short exposure of the 2F12 blot are shown. <b>C.</b> Cytosols (post-100,000 <i>g</i>) of SH-SY5Y cells were crosslinked with 2 mM DSP, followed by boiling in sample buffer/5% βME and blotting for the indicated antibodies. Membranes were cut at dotted lines after protein transfer and the left half was incubated in 0.4% PFA for 30 min, while the right half was blocked immediately. Membranes were reassembled before film development; PFA-treated and -untreated halves shown were exposed identically. <b>D.</b> M17D cells analyzed analogously to SH-SY5Y cells in Fig. 4c. In addition to αSyn and UCH-L1, histone protein H3 was detected using a total H3 as well as an H3 lysine 27-methylation-specific antibody (H3 K27met).</p

Discrepancy between immunoblotting of αSyn from crosslinker-treated and untreated lysates.

<p><b>A.</b> Intact primary cortical rat neurons (13 days <i>in vitro</i>) were incubated with DMSO only (-) or a gradient of 0.1, 0.5 and 1.0 mM DSP or DSG, sonicated in PBS supplemented with protease inhibitors and spun at 100,000 <i>g</i> for 60 min. The resultant cytosols (30 µg) were blotted with αSyn antibody Syn1 (left panel: short exposure; middle panel: long exposure) and with anti-DJ-1 as a control (right panel). Arrows indicate the crosslinker-trapped αSyn species that have been described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081314#pone.0081314-Dettmer1" target="_blank">[7]</a> as well as the DJ-1 dimer (2-mer) and monomer (1-mer). <b>B.</b> Densitometric analysis of the blots shown in Fig. 1a using ImageJ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081314#s2" target="_blank">Materials and Methods</a>). Bars indicate the total immunoreactivity of each lane as a percentage of the signal from DMSO-only treated sample (lane 1), after subtraction of the background from the empty lane 5.</p

Additional file 1: of Decoding the synaptic dysfunction of bioactive human AD brain soluble Aβ to inspire novel therapeutic avenues for Alzheimer’s disease

Table S1. Demographic and pathological data on brain samples. Figure S1. Characterization of AD brain extracts used for LTP experiments. (a) Half milliliter aliquots of mock immunodepleted (AD) and AW7 immunodepleted (ID-AD) extracts were analyzed by IP/WB. AW7 was used for IP and a combination of 2G3 and 21F12 was used for WB. To enable comparison 2 and 5 ng of Aβ1–42 peptide was also electrophoresed on the gel. IP/WB analysis allows the capture of Aβ structures under native conditions and their detection following denaturing SDS-PAGE. (b) The same samples were also analyzed by an MSD-based Aβx-42 immunoassay. Since GuHCl effectively disaggregates high molecular weight Aβ species, samples were analyzed with and without incubation in denaturant. Analysis of samples in the absence of GuHCl allows the measurement of native Aβ monomer, whereas, analysis of samples treated with GuHCl allows detection of disassembled aggregates. The AD extracts contained much larger amounts of aggregates than monomer, and both monomer and aggregates were effectively removed by AW7 immunodepletion. The experiments shown are typical of at least 3 separate experiments. Figure S2. Bath application of anti-Aβ antibodies had no significant effect on hippocampal LTP. Each data in this graph was average of at least 6 recordings. (DOCX 466 kb

Discovery of a Novel Pharmacological and Structural Class of Gamma Secretase Modulators Derived from the Extract of Actaea racemosa

A screen of a library of synthetic drugs and natural product extracts identified a botanical extract that modulates the processing of amyloid precursor protein (APP) in cultured cells to produce a lowered ratio of amyloid-beta peptide (1–42) (Aβ42) relative to Aβ40. This profile is of interest as a potential treatment for Alzheimer’s disease. The extract, from the black cohosh plant (Actaea racemosa), was subjected to bioassay guided fractionation to isolate active components. Using a combination of normal-phase and reverse-phase chromatography, a novel triterpene monoglycoside, <b>1</b>, was isolated. This compound was found to have an IC₅₀ of 100 nM for selectively reducing the production of amyloidogenic Aβ42 while having a much smaller effect on the production of Aβ40 (IC₅₀ 6.3 μM) in cultured cells overexpressing APP. Using IP-MS methods, this compound was found to modulate the pool of total Aβ produced by reducing the proportion of Aβ42 while increasing the relative amounts of shorter and less amyloidogenic Aβ37 and Aβ39. Concentrations of <b>1</b> sufficient to lower levels of Aβ42 substantially (up to 10 μM) did not significantly affect the processing of Notch or other aspects of APP processing. When <b>1</b> (10 μg) was administered to CD-1 normal mice intracerebroventricularly, the level of Aβ42 in brain was reduced. Assays for off-target pharmacology and the absence of overt signs of toxicity in mice dosed with compound <b>1</b> suggest a comparatively selective pharmacology for this triterpenoid. Compound <b>1</b> represents a new lead for the development of potential treatments for Alzheimer’s disease via modulation of gamma-secretase