Amyloid-Beta (Aβ) D7H Mutation Increases Oligomeric Aβ42 and Alters Properties of Aβ-Zinc/Copper Assemblies

Amyloid precursor protein (APP) mutations associated with familial Alzheimer's disease (AD) usually lead to increases in amyloid β-protein (Aβ) levels or aggregation. Here, we identified a novel APP mutation, located within the Aβ sequence (AβD7H), in a Taiwanese family with early onset AD and explored the pathogenicity of this mutation. Cellular and biochemical analysis reveal that this mutation increased Aβ production, Aβ42/40 ratio and prolonged Aβ42 oligomer state with higher neurotoxicity. Because the D7H mutant Aβ has an additional metal ion-coordinating residue, histidine, we speculate that this mutation may promote susceptibility of Aβ to ion. When co-incubated with Zn2+ or Cu2+, AβD7H aggregated into low molecular weight oligomers. Together, the D7H mutation could contribute to AD pathology through a “double punch” effect on elevating both Aβ production and oligomerization. Although the pathogenic nature of this mutation needs further confirmation, our findings suggest that the Aβ N-terminal region potentially modulates APP processing and Aβ aggregation, and further provides a genetic indication of the importance of Zn2+ and Cu2+ in the etiology of AD

Characterization of Protein Hydrolysates from Eel (Anguilla marmorata) and Their Application in Herbal Eel Extracts

The enzymatic hydrolysis of fish proteins is the principle method for converting under-utilized fish into valuable products for the pharmaceutical and health food industries. In this study, three commercial enzymes (alcalase, bromelain, and papain) were tested for their ability to create eel protein hydrolysates (EPHs) from whole eel (Anguilla marmorata). Freeze-dried EPHs had almost more than 80% solubility (p < 0.05) in solutions ranging from pH 2–10. The amino acid profiles of the EPHs showed a high percentage of essential amino acids, including histidine, threonine, valine, isoleucine, and leucine. The emulsion activity index (EAI) of EPH resulted as follows: alcalase group (36.8 ± 2.00) > bromelain group (21.3 ± 1.30) > papain group (16.2 ± 1.22), and the emulsion stability index (ESI) of EPH was: alcalase group (4.00 ± 0.34) > bromelain group (2.62 ± 0.44) > papain group (1.44 ± 0.09). As such, EPH has a high nutritional value and could be used as a supplement to diets lacking protein. EPH showed excellent solubility and processed interfacial properties, which are governed by its concentration. Among of them the alcalase group had the best antioxidant effect at 1,1-diphenyl-2-pyridinohydrazinyl (DPPH) radical method, determination of reducing power and ABTS test compared with other groups. EPH may be useful in developing commercial products like herbal eel extracts that are beneficial to human health

A small/wide-angle X-ray scattering instrument for structural characterization of air-liquid interfaces, thin films and bulk specimens

At the National Synchrotron Radiation Research Center, a small/wide-angle X-ray scattering (SAXS/WAXS) instrument has been installed at the BL23A beamline with a superconducting wiggler insertion device. This beamline is equipped with double Si(111) crystal and double Mo/B4C multilayer monochromators, and an Si-based plane mirror that can selectively deflect the beam downwards for grazing-incidence SAXS (GISAXS) studies of air-liquid or liquid-liquid interfaces. The SAXS/WAXS instrument, situated in an experimental hutch, comprises collimation, sample and post-sample stages. Pinholes and slits have been incorporated into the beam collimation system spanning a distance of ̃5 m. The sample stage can accommodate various sample geometries for air-liquid interfaces, thin films, and solution and solid samples. The post-sample section consists of a 1 m WAXS section with two linear gas detectors, a vacuum bellows (1-4 m), a two-beamstop system and the SAXS detector system, all situated on a motorized optical bench for motion in six degrees of freedom. In particular, the vacuum bellows of a large inner diameter (260 mm) provides continuous changes of the sample-to-detector distance under vacuum. Synchronized SAXS and WAXS measurements are realized via a data-acquisition protocol that can integrate the two linear gas detectors for WAXS and the area detector for SAXS (gas type or Mar165 CCD); the protocol also incorporates sample changing and temperature control for programmable data collection. The performance of the instrument is illustrated via several different measurements, including (1) simultaneous SAXS/WAXS and differential scanning calorimetry for polymer crystallization, (2) structural evolution with a large ordering spacing of ̃250 nm in a supramolecular complex, (3) SAXS for polymer blends under in situ drawing, (4) SAXS and anomalous SAXS for unilamellar lipid vesicles and metalloprotein solutions, (5) anomalous GISAXS for oriented membranes of Br-labeled lipids embedded with peptides, and (6) GISAXS for silicate films formed in situ at the air-water interface

The D7H mutation decreases the redox activity of Aβ42 in metal reduction assay.

<p>Reduction of Cu²⁺ to Cu⁺ was performed by BCA assay. Freshly prepared 10 μM Aβ42_wt and Aβ42_D7H were mixed with BCA solution containing 4% CuSO₄ to perform the redox activity assay. Data were presented as mean ± SEM (n = 3), ***<i>P</i><0.0001.</p

Aβ morphology in the presence or absence of metal ions was revealed by TEM.

<p>Lyophilized Aβ was prepared in HFIP-DMSO. After 264–312 h of incubation in either the presence or absence of Zn²⁺ or Cu²⁺, the Aβ samples were stained by 2% uranyl acetate and monitored by TEM. In the presence of ions, the Aβ_D7H peptides were predominantly amorphous morphology but not protofibrils as Aβ_wt. Scale bar: 200 nm.</p

The D7H mutation enhances the neurotoxicity of Aβ42.

<p>The neurotoxicities of Aβ42_wt and Aβ42_D7H were estimated by the MTT assay. SH-SY5Y cells were treated with Aβ42_wt or Aβ42_D7H at a final concentration of 0, 5, or 10 μM for 48 h. Cell survival was determined by normalizing OD570 readings to those of cells not treated with Aβ42 (set as 1) in 3 independent experiments (n = 8 per experiment) and is presented as mean ± SEM. ***<i>P</i><0.001, **<i>P</i><0.01 vs. Aβ42_wt by ANOVA.</p

A novel mutation leads to an aspartate to histidine substitution at the N-terminus of Aβ.

<p>(A) The upper part of the diagram presents the Aβ42 sequence with the location of the D7H mutation (red). As shown in the lower part of the diagram, processing of APP occurs via two pathways. Nonamyloidogenic processing of APP by á-secretase produces the C83 and sAPPα fragments; amyloidogenic processing of APP by â-secretase produces the C99 and sAPPβ fragments. Aβ is generated through subsequent cleavage of C99 by γ-secretase. (B) SPECT images of the index patient indicate hypoperfusion in the bilateral parietal cortices and the left temporal cortex. (C) Direct sequencing of APP exon 16 PCR products derived from the patient and from healthy controls revealed a GAC-to-CAC nucleotide substitution in Aβ region of the patient's APP gene (in 678^th amino acid using APP770 numbering or in 7^th amino acid using Aβ numbering).</p

The D7H mutation shifts Zn²⁺ and Cu²⁺-induced assemblies toward smaller oligomers with fewer fibrils.

<p>(A–H) 25 μM Aβ was incubated with 25 μM ThT in Tris buffer containing 0 μM (black), 5 μM (light color), 12.5 μM (medium color) or 25 μM (dark color) of Zn²⁺ (red) or Cu²⁺ (blue). (A) Aβ40_wt + Zn²⁺, (B) Aβ40_wt + Cu²⁺, (C) Aβ40_D7H + Zn²⁺, (D) Aβ40_D7H + Cu²⁺, (E) Aβ42_wt + Zn²⁺, (F) Aβ42_wt + Cu²⁺, (G) Aβ42_D7H + Zn²⁺, (H) Aβ42_D7H + Cu²⁺. (I–J) 25 μM Aβ40 (I) and Aβ42 (J) were co-incubated with 25 μM Zn²⁺ or Cu²⁺ for 114 h, fixed by PICUP and examined by Western blot to analyze the size distribution.</p