Amyloid-Beta Protein Clearance and Degradation (ABCD) Pathways and their Role in Alzheimer’s Disease

Amyloid-β proteins (Aβ) of 42 (Aβ42) and 40 aa (Aβ40) accumulate as senile plaques (SP) and cerebrovascular amyloid protein deposits that are defining diagnostic features of Alzheimer's disease (AD). A number of rare mutations linked to familial AD (FAD) on the Aβ precursor protein (APP), Presenilin-1 (PS1), Presenilin- 2 (PS2), Adamalysin10, and other genetic risk factors for sporadic AD such as the ε4 allele of Apolipoprotein E (ApoE-ε4) foster the accumulation of Aβ and also induce the entire spectrum of pathology associated with the disease. Aβ accumulation is therefore a key pathological event and a prime target for the prevention and treatment of AD. APP is sequentially processed by β-site APP cleaving enzyme (BACE1) and γ-secretase, a multisubunit PS1/PS2-containing integral membrane protease, to generate Aβ. Although Aβ accumulates in all forms of AD, the only pathways known to be affected in FAD increase Aβ production by APP gene duplication or via base substitutions on APP and γ-secretase subunits PS1 and PS2 that either specifically increase the yield of the longer Aβ42 or both Aβ40 and Aβ42. However, the vast majority of AD patients accumulate Aβ without these known mutations. This led to proposals that impairment of Aβ degradation or clearance may play a key role in AD pathogenesis. Several candidate enzymes, including Insulin-degrading enzyme (IDE), Neprilysin (NEP), Endothelin-converting enzyme (ECE), Angiotensin converting enzyme (ACE), Plasmin, and Matrix metalloproteinases (MMPs) have been identified and some have even been successfully evaluated in animal models. Several studies also have demonstrated the capacity of γ-secretase inhibitors to paradoxically increase the yield of Aβ and we have recently established that the mechanism is by skirting Aβ degradation. This review outlines major cellular pathways of Aβ degradation to provide a basis for future efforts to fully characterize the panel of pathways responsible for Aβ turnover

New evidences on Tau-DNA interactions and relevance to neurodegeneration

Tau is mainly distributed in cytoplasm and also found to be localized in the nucleus. There is limited data on DNA binding potential of Tau.We provide novel evidence on nicking of DNA by Tau. Tau nicks the supercoiled DNA leading to open circular and linear forms. The metal ion magnesium (a co-factor for endonuclease) enhanced the Tau DNA nicking ability, while an endonuclease specific inhibitor,aurinetricarboxylic acid (ATA) inhibited the Tau DNA nicking ability Further, we also evidenced that Tau induces B-C-A mixed conformational transition in DNA and also changes DNA stability. Tau-scDNA complex is more sensitive to DNAse I digestion indicating stability changes in DNA caused by Tau. These findings indicate that Tau alters DNA helicity and integrity and also nicks the DNA. The relevance of these novel intriguing findings regarding the role Tau in neuronal dysfunction is discussed. (C) 2010 Elsevier Ltd. All rights reserved

In vitro Evidence that an Aqueous Extract of Centella asiatica Modulates alpha-Synuclein Aggregation Dynamics

alpha-Synuclein aggregation is one of the major etiological factors implicated in Parkinson's disease (PD). The prevention of aggregation of alpha-synuclein is a potential therapeutic intervention for preventing PD. The discovery of natural products as alternative drugs to treat PD and related disorders is a current trend. The aqueous extract of Centella asiatica (CA) is traditionally used as a brain tonic and CA is known to improve cognition and memory. There are limited data on the role of CA in modulating amyloid-beta (A beta) levels in the brain and in A beta aggregation. Our study focuses on CA as a modulator of the alpha-synuclein aggregation pattern in vitro. Our investigation is focused on: (i) whether the CA leaf aqueous extract prevents the formation of aggregates from monomers (Phase I: alpha-synuclein + extract co-incubation); (ii) whether the CA aqueous extract prevents the formation of fibrils from oligomers (Phase II: extract added after oligomers formation); and (iii) whether the CA aqueous extract disintegrates the pre-formed fibrils (Phase III: extract added to mature fibrils and incubated for 9 days). The aggregation kinetics are studied using a thioflavin-T assay, circular dichroism, and transmission electron microscopy. The results showed that the CA aqueous extract completely inhibited the alpha-synuclein aggregation from monomers. Further, CA extract significantly inhibited the formation of oligomer to aggregates and favored the disintegration of the preformed fibrils. The study provides an insight in finding new natural products for future PD therapeutics

Evidence of a novel mechanism for partial γ-secretase inhibition induced paradoxical increase in secreted amyloid β protein.

BACE1 (β-secretase) and α-secretase cleave the Alzheimer's amyloid β protein (Aβ) precursor (APP) to C-terminal fragments of 99 aa (CTFβ) and 83 aa (CTFα), respectively, which are further cleaved by γ-secretase to eventually secrete Aβ and Aα (a.k.a. P3) that terminate predominantly at residues 40 and 42. A number of γ-secretase inhibitors (GSIs), such as N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), have been developed with the goal of reducing Aβ to treat Alzheimer's disease (AD). Although most studies show that DAPT inhibits Aβ in a dose-dependent manner several studies have also detected a biphasic effect with an unexpected increase at low doses of DAPT in cell cultures, animal models and clinical trials. In this article, we confirm the increase in Aβ40 and Aβ42 in SH-SY5Y human neuroblastoma cells treated with low doses of DAPT and identify one of the mechanisms for this paradox. We studied the pathway by first demonstrating that stimulation of Aβ, a product of γ-secretase, was accompanied by a parallel increase of its substrate CTFβ, thereby demonstrating that the inhibitor was not anomalously stimulating enzyme activity at low levels. Secondly, we have demonstrated that inhibition of an Aβ degrading activity, endothelin converting enzyme (ECE), yielded more Aβ, but abolished the DAPT-induced stimulation. Finally, we have demonstrated that Aα, which is generated in the secretory pathway before endocytosis, is not subject to the DAPT-mediated stimulation. We therefore conclude that impairment of γ-secretase can paradoxically increase Aβ by transiently skirting Aβ degradation in the endosome. This study adds to the growing body of literature suggesting that preserving γ-secretase activity, rather than inhibiting it, is important for prevention of neurodegeneration

PA treatment mitigates Aβ stimulation.

<p>SH-SY5Y cells were treated with a combination of PA and DAPT (Blue squares and dashed lines) or DAPT alone (Red circles, continuous line) and Aβ40 (A) or Aβ42 (C) were plotted as raw data in pg/ml or as percent change (C, D). The change from the 0 DAPT control show that stimulation by low level GSI treatment is completely blocked by PA treatment for Aβ40 (B) but remained at 1.4 fold for Aβ42 (D). Nevertheless, the DAPT-induced stimulation of Aβ42 at 12.5 and 25 nM was also significantly (p = 0.03) attenuated (C, D). The DAPT-mediated stimulation in PA-treated cells is 4-fold for Aβ40 compared to two fold for Aβ42, suggesting that ECE contributes more substantially to Aβ40 turnover than Aβ42, but DAPT increases Aβ42 equally by avoiding other unidentified degrading enzymes by the same mechanism.</p

The secreted APP fragment generated by α and γ secretase is not stimulated by DAPT.

<p>SH-SY5Y cells were treated with a combination of DAPT and media were analyzed using a combination of ELISA assays capturing secreted fragments ending at Aβ residue 40 and 42 and detecting with 4G8 to determine the total Ax40 and Ax42 (red circles, solid line) and the Aβ40 and Aβ42 values were subtracted to obtain Aα40 and Aα42 (blue triangles, dotted lines). Although similar trends were observed for Ax42 and Ax42 Tukey-Kramer analysis gave highly significant P values for increase in Ax40 (<0.05; A) but not for Ax42 (>0.7). (<0.0002; B). In contrast Aα40 showed a dose-dependent inhibition (p<0.0001) for all doses except 12.5 nM (p = 0.054) with no stimulation at 12.5 and 25 nM DAPT. Aα42 showed similar trends, but the values were not significant. Furthermore, elimination of an outlier control revealed a small, but significant increase in Aα42 (not shown).</p

PA treatment does not affect APP processing.

<p>SH-SY5Y-APP695 cells were treated with 0 to 1,000 nM DAPT in the absence and presence of 100 μM PA and cell lysates were analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091531#pone-0091531-g003" target="_blank">Fig 3</a>. Panel A is a representative Western blot with O443 showing APP (top band), CTFβ (Faint lower band) and CTFα (dark lowest band). The quantified band intensities for APP (B), CTFα (C) and CTFβ (D) show the changes observed with DAPT treatment in the presence (blue bar and dotted lines with solid diamonds) or absence of PA (red bars, solid red circles). Note that the labels on the x-axis are not drawn to scale to include the 1000 nM DAPT data. Tukey-Kramer analysis show that there is significant dose-dependent increase in CTFβ (p<0.002) and CTFα (p<0.0001) with DAPT treatment, but there is no significant change in either CTFβ (p>0.2, 0.5) or CTFα (p>0.8, 0.9).</p

Key APP processing pathways.

<p>APP is a type-1 integral-membrane glycoprotein with a large ectodomain a single transmembrane domain and a short intracellular domain (B). While it exists in multiple forms, we are using neuroblastoma cells overexpressing the neuronal 695 aa form. In this form, the ectodomain includes a region of 596 aa that is cleaved and secreted by BACE1 called sAPPβ (Blue ellipse) leaving behind the CTFβ of 99 aa (C). The Aβ sequence starts with the first 16 aa, which is released with the 596 aa after cleavage by α-secretase to sAPPα of 612 aa, and CTFα of 83 aa (A). The presenilin-containing multisubunit γ-secretase cleaves CTFα (A) and CTFβ (C) to secreted proteins of 3 kDa (Aα) and 4 kDa (Aβ), respectively. Although multiple intramembrane intermediate forms of Aβ and P3 are reported, the major secreted forms terminate at residue 40 followed at much lower levels by residue 42 of the Aβ sequence. Numerous studies have demonstrated that most FAD mutations preferentially increase Aβ42. Antibodies used in the study are indicated above the APP schematic (B). Domains of APP are not drawn to scale but colors are consistent in all figures. Note that we are using Aα instead of the more common P3 or Aβ 17–40/42 to maintain processing pathway consistency that makes it easier for the non-expert.</p

CHO cells fail to display the DAPT induced stimulation of Aβ.

<p>CHO cells and SH-SY5Y cells expressing human APP695 were treated with 6.25, 12.5, 25, 50 and 100 nM DAPT for 2 h. DAPT induced the consistent increase in Aβ40 at 6.25 nM (<0.05), but the CHO-APP695 failed to show any stimulation of Aβ40 but showed a trend towards reduction (P = 0.12) instead. Aβ42 was not detectable in these transfected CHO- APP695 cells.</p

Model showing the mechanism for increase in Aβ with γ-secretase impairment.

<p>The hypothetical model derives from the known cellular localization of Aβ production and turnover pathways to explain the unexpected and contrasting changes in APP metabolites with DAPT treatment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091531#pone.0091531-Sambamurti3" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091531#pone.0091531-Selkoe1" target="_blank">[78]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091531#pone.0091531-Choy1" target="_blank">[80]</a>. APP shown with a black circle to the β-secretase site, an open circle to represent Aβ1-16 to the junction of the α-secretase cleavage site and a grey circle for the remainder of Aβ starting at position 17. Secreted sAPPα is shown as a joined black and open circle, Aβ as an open and grey circle, Aα as a grey circle and CTFα/β as a combination of circles with a tail embedded in the membrane. APP is predominantly processed by α-secretase in the secretory pathway from the trans-Golgi network (TGN) to the cell surface generating CTFα, which is processed by γ-secretase in the secretory pathway (A). On the other hand, BACE1 cleaves APP in the endocytosis pathway to C99 where γ-secretase generates some Aβ (D). The Aβ (B) and remaining unprocessed C99 (C) are transported to the surface where the residual C99 is converted to Aβ by γ-secretase and both pools of Aβ are secreted into the medium. Aβ, but not C99 is degraded primarily by ECE in the endosome (D, E). Inhibition by GSI increases the C99 pool in the endosome (E) but this C99 is further processed during the recycling step where it reaches the cell surface either directly or via the TGN to generate Aβ that now escapes degradation in the endosome. One can also increase the secreted Aβ by inhibiting ECE with PA, but this increase does not affect CTFβ (F).</p