Cytoplasmic Fragment of Alcadein alpha Generated by Regulated Intramembrane Proteolysis Enhances Amyloid beta-Protein Precursor (APP) Transport into the Late Secretory Pathway and Facilitates APP Cleavage

The neural type I membrane protein Alcadein alpha (Alc alpha), is primarily cleaved by amyloid beta-protein precursor (APP) alpha-secretase to generate a membrane-associated carboxyl-terminal fragment (Alc alpha CTF), which is further cleaved by gamma-secretase to secrete p3-Alc alpha peptides and generate an intracellular cytoplasmic domain fragment (Alc alpha ICD) in the late secretory pathway. By association with the neural adaptor protein X11L (X11-like), Alc alpha and APP form a ternary complex that suppresses the cleavage of both Alc alpha and APP by regulating the transport of these membrane proteins into the late secretory pathway where secretases are active. However, it has not been revealed how Alc alpha and APP are directed from the ternary complex formed largely in the Golgi into the late secretory pathway to reach a nerve terminus. Using a novel transgenic mouse line expressing excess amounts of human Alc alpha CTF (hAlc alpha CTF) in neurons, we found that expression of hAlc alpha CTF induced excess production of hAlc alpha ICD, which facilitated APP transport into the nerve terminus and enhanced APP metabolism, including A beta generation. In vitro cell studies also demonstrated that excess expression of Alc alpha ICD released both APP and Alc alpha from the ternary complex. These results indicate that regulated intramembrane proteolysis of Alc alpha by gamma-secretase regulates APP trafficking and the production of A beta in vivo

Mechanism of Intramembrane Cleavage of Alcadeins by γ-Secretase

<div><p>Background</p><p>Alcadein proteins (Alcs; Alcα, Alcβand Alcγ) are predominantly expressed in neurons, as is Alzheimer's β-amyloid (Aβ) precursor protein (APP). Both Alcs and APP are cleaved by primary α- or β-secretase to generate membrane-associated C-terminal fragments (CTFs). Alc CTFs are further cleaved by γ-secretase to secrete p3-Alc peptide along with the release of intracellular domain fragment (Alc ICD) from the membrane. In the case of APP, APP CTFβ is initially cleaved at the ε-site to release the intracellular domain fragment (AICD) and consequently the γ-site is determined, by which Aβ generates. The initial ε-site is thought to define the final γ-site position, which determines whether Aβ40/43 or Aβ42 is generated. However, initial intracellular ε-cleavage sites of Alc CTF to generate Alc ICD and the molecular mechanism that final γ-site position is determined remains unclear in Alcs.</p><p>Methodology</p><p>Using HEK293 cells expressing Alcs plus presenilin 1 (PS1, a catalytic unit of γ-secretase) and the membrane fractions of these cells, the generation of p3-Alc possessing C-terminal γ-cleavage site and Alc ICD possessing N-terminal ε-cleavage site were analysed with MALDI-TOF/MS. We determined the initial ε-site position of all Alcα, Alcβ and Alcγ, and analyzed the relationship between the initially determined ε-site position and the final γ-cleavage position.</p><p>Conclusions</p><p>The initial ε-site position does not always determine the final γ-cleavage position in Alcs, which differed from APP. No additional γ-cleavage sites are generated from artificial/non-physiological positions of ε-cleavage for Alcs, while the artificial ε-cleavage positions can influence in selection of physiological γ-site positions. Because alteration of γ-secretase activity is thought to be a pathogenesis of sporadic Alzheimer's disease, Alcs are useful and sensitive substrate to detect the altered cleavage of substrates by γ-secretase, which may be induced by malfunction of γ-secretase itself or changes of membrane environment for enzymatic reaction.</p></div

Alteration of Alcs γ-cleavage when physiological ε-cleavage sites are replaced with non-physiological/pseudo-ε-cleavage sites.

<p>A. Positions of the physiological major and minor (ε1 and ε2) and pseudo- (ε1p and ε2p) ε-cleavage sites (upper left) are shown along with the physiological major and minor γ-cleavage sites (γ1 and γ2). The shaded amino acid sequence indicates a putative membrane-embedded region. Non-physiological/pseudo-ε-cleavage sites were designed by shifting one residue toward the N terminal of the physiological ε-cleavage sites. Representative MS spectra of p3-Alcα secreted by HEK293 cells expressing Alcα CTF, Alcα CTF-ε1, Alcα CTF-ε2, Alcα CTF-ε1p, or Alcα CTF-ε2p are shown (lower left panels). The major species p3-Alcα2N+35 with γ1 site (γ1/35, closed arrowheads) and minor species p3-Alcα2N+38 with γ2 site (γ2/38, open arrowheads) are indicated. The peak area of p3-Alcα2N+38 was compared with that of p3-Alcα2N+35, and the ratios (p3-Alcα2N+38/p3-Alcα2N+35) are indicated as γ2/γ1 (right panel). The spectra of minor species p3-Alcα38 are enlarged in windows in which intensities of 200, 300, and 400 on the y-axis correspond to 0.02, 0.03 and 0.04 in the original panels. <b>B.</b> Positions of the physiological major and minor (ε1, ε2, and ε3) and pseudo- (ε1p, ε2p, and ε3p) ε-cleavage sites (upper left) are shown along with the physiological major and minor γ-cleavage sites (γ1 and γ2). Representative MS spectra of p3-Alcβ secreted by HEK293 cells expressing Alcβ CTF, Alcβ CTF-ε1, Alcβ CTF-ε2, Alcβ CTF-ε3, Alcβ CTF-ε1p, Alcβ CTF-ε2p, or Alcβ CTF-ε3p are shown (lower left). The major species p3-Alcβ40 withγ1 site (γ1/40, closed arrowheads) and minor species p3-Alcβ37 with γ2 site (γ2/37, open arrowheads) are indicated. The peak area of p3-Alcβ37 was compared with that of p3-Alcβ40, and the ratios (p3-Alcβ37/p3-Alcβ40) are indicated asγ2/γ1 (right panel). <b>C.</b> Positions of the physiological major and minor (ε1, ε2, and ε3) and pseudo- (ε1p, ε2p, and ε3p) ε-cleavage sites (upper left) are shown along with the physiological major and minor γ-cleavage sites (γ1 and γ2). Representative MS spectra of p3-Alcγ secreted by HEK293 cells expressing Alcγ CTF, Alcγ CTF-ε1, Alcγ CTF-ε2, Alcγ CTF-ε3, Alcγ CTF-ε1p, Alcγ CTF-ε2p, or Alcγ CTF-ε3p are shown (lower left). The major p3-Alcγ31 with γ1 site (γ1/31, closed arrowhead) and minor p3-Alcγ34 with γ2 site (γ2/34, open arrowhead) are indicated. The peak area of p3-Alcγ34 was compared with that of p3-Alcγ31, and the ratios (p3-Alcγ34/p3-Alcγ31) are indicated as γ2/γ1 (right panel). (<b>A–C</b>) The ratios of products from the pseudo-site were compared to those from the respective physiological sites. Statistical analysis was performed by Student's t test (mean ± S.E., n = 4, *<i>P</i><0.05).</p

Alteration of ε-cleavage is not necessarily prerequisite to determine a specific γ-cleavage site in Alcα.

<p>A<b>.</b> Representative MS spectra of p3-Alcα secreted by HEK293 cells expressing Alcα CTF, Alcα CTF-ε1, or Alcα CTF-ε2 with either wild-type PS1 (wt) or a FAD-linked PS1 mutant (A434C, L166P, or R278T). The p3-Alcα species in cell culture media were immunoprecipitated and subjected to MALDI-TOF/MS analysis. Closed arrowheads indicate the major product with γ1 site (p3-Alcα2N+35, “γ1/35”), while open arrowheads indicate the minor product with γ2 site (p3-Alcα2N+38, “γ2/38”). The spectra of the minor p3-Alcα38 product are enlarged in windows in which intensity of 300 on the y-axis corresponds to 0.03 in the original panels. <b>B.</b> The peak area of p3-Alcα2N+38 (minor species) was compared with that of p3-Alcα2N+35 (major species), and the minor to major ratios (p3-Alcα2N+38/p3-Alcα2N+35) are indicated asγ2/γ1. Statistical analysis was performed using one-way analysis of variance followed by the Tukey-Kramer multiple comparison test (means ± S.E., n = 4). Significance in comparison to the ratio of Alcα CTF was not observed in cells expressing wild-type PS1 (wt) or FAD-linked PS1 mutants. The columns of “wt” and “A434C” are enlarged in window.</p

Determination of intramembrane ε-cleavage sites of Alcadeins.

<p>Representative mass spectra of Alc ICD-ΔC-FLAG generated by <i>in vitro</i> γ-secretase assay with membranes from HEK293 cells expressing Alc-ΔC-FLAG (<b>A</b>), and localization of ε-cleavage sites on the amino acid sequence (<b>B</b>), along with the comparison to γ- and ε-cleavage sites of APP (<b>C</b>). <b>A.</b> AlcαICD-ΔC-FLAG generated from Alcα-ΔC-FLAG (left), Alcβ ICD-ΔC-FLAG generated from Alcβ-ΔC-FLAG (middle), and Alcγ ICD-ΔC-FLAG generated from Alcγ-ΔC-FLAG (right). Closed arrowheads indicate the major product cleaved at the ε1 site, and open arrowheads indicate the minor products cleaved at the ε2 and ε3 sites. Amino acid sequence of Alc ICD-ΔC-FLAG was determined (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062431#pone.0062431.s003" target="_blank">Fig. S3</a></b>). Other peaks, which are not indicated with arrowheads, are not products derived from Alc-ΔC-FLAG, because they are detectable in cells expressing an inactive/dominant-negative PS1 D385A mutant (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062431#pone.0062431.s002" target="_blank">Fig. S2</a></b>). <b>B.</b> Amino acid sequence of human Alcα1, Alcβ and Alcγ (numbers indicate amino acid position, and the broken underline indicates putative transmembrane region). The major (ε1) and minor (ε2 and ε3) ε-cleavage sites are indicated, along with the previously identified major (γ1) and minor (γ2) γ-cleavage sites (13). In Alcα (upper), cleavage at γ1 generates p3-Alcα35, while cleavage at γ2 generates p3-Alcα38. Therefore, the γ1 cleavage site “γ1/35” and the γ2 cleavage site “γ2/38” are shown. Cultured cell lines generate p3-Alcα2N+35 and p3-Alcα2N+38, which possess two extra amino acids at the N terminal but have identical γ-cleavage sites to p3-Alcα35 and p3-Alcα38 in human CSF and are therefore considered the products cleaved at γ1 and γ2, respectively. In Alcβ (middle), cleavage at γ1 (γ1/40) generates p3-Alcβ40, while cleavage at γ2 (γ2/37) generates p3-Alcβ37. In cultured cell lines, “γ1/40” is the major γ-cleavage site, but “γ2/37” is the major site in human CSF. In Alcγ (lower), cleavage at the major cleavage siteγ1 (γ1/31) generates p3-Alcγ31, while cleavage at the minor site γ2 (γ2/34) generates p3-Alcγ34. Schematic pictures of protein constructs used in this study are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062431#pone.0062431.s001" target="_blank">Fig. S1</a>. </b><b>C.</b> Major and minor γ- and ε-cleavage sites of APP. The major γ1 (γ1/40) and minor γ2 (γ2/42) cleavage sites are shown. In APP, the major ε1 site largely defines the γ1 site to generate Aβ40, and the minor ε2 site promotes the γ2 site to generate Aβ42 or Aβ38.</p

Magnitudes of the minor to major ratios of γ-cleavage and ε-cleavage in Alcs and APP from cells expressing FAD-linked PS1 mutants.

<p>Minor to major p3-Alc ratio (γ2/γ1, first rows) was determined by quantitative MS analysis of p3-Alc secreted by HEK293 cells expressing Alc-ΔC-FLAG with either wild-type PS1 or PS1 carrying a FAD-linked mutation (A434C, L166P, or R278T) (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062431#pone.0062431.s005" target="_blank">Fig. S5</a></b>). Minor to major Alc ICD ratios (ε2/ε1, second rows; ε3/ε1, third rows) were also determined by MS analysis of Alc ICD-ΔC-FLAG generated by <i>in vitro</i> γ-secretase assay with membranes from the same cells (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062431#pone.0062431.s005" target="_blank">Fig. S5</a></b>). <b>A. Comparison of Alcα γ-cleavage ratio to ε-cleavage ratio.</b> The ratio (γ2/γ1) of p3-Alcα2N+38 (minor) to p3-Alcα2N+35(major) (first row) and the ratio (ε2/ε1) of the Alcα ICD-ΔC-FLAG product cleaved at ε2 (minor) to the product cleaved at ε1 (major) (second row) are shown. <b>B. Comparison of Alcβ γ-cleavage ratio to ε-cleavage ratio.</b> The ratio (γ2/γ1) of p3-Alcβ37 (minor) to p3-Alcβ40 (major) (first row) and the ratio (ε2/ε1 or ε3/ε1) of the Alcβ ICD-ΔC-FLAG product cleaved at ε2 or ε3 (minor) to the product cleaved at ε1 (major) (second row, ε2/ε1; third row, ε3/ε1) are shown. <b>C. Comparison of Alcγ γ-cleavage ratio to ε-cleavage ratio.</b> The ratio (γ2/γ1) of p3-Alcγ34 (minor) to p3-Alcγ31 (major) (first row) and the ratio (ε2/ε1 or ε3/ε1) of the Alcγ ICD-ΔC-FLAG product cleaved at ε2 or ε3 (minor) to the product cleaved at ε1 (major) (second row, ε2/ε1; third row, ε3/ε1) are shown. <b>D. Comparison of APP γ-cleavage ratio to ε-cleavage ratio.</b> The ratio (γ2/γ1) of Aβ42 (minor) to Aβ40 (major) (first row) and the minor to major ratio of AICD (second row, ε2/ε1) are shown. Net Aβ values are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062431#pone.0062431.s012" target="_blank">Table S2</a></b>, and representative mass spectra of AICD-FLAG are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062431#pone.0062431.s005" target="_blank">Fig. S5D</a></b>. Statistical analysis was performed using Dunnett’s multiple comparison test. Significance is indicated relative to the ratio of wild-type PS1 (wt) (mean ± S.E., n = 4, *<i>P</i><0.05, **<i>P</i><0.001, ***<i>P</i><0.0001).</p

Proportion and clinical characteristics of non-asthmatic non-smokers among adults with airflow obstruction

<div><p>Background and objectives</p><p>Chronic obstructive pulmonary disease (COPD) mainly develops after long-term exposure to cigarette or biomass fuel smoke, but also occurs in non-smokers with or without a history of asthma. We investigated the proportion and clinical characteristics of non-smokers among middle-aged to elderly subjects with airflow obstruction.</p><p>Methods</p><p>We retrospectively analyzed 1,892 subjects aged 40–89 years who underwent routine preoperative spirometry at a tertiary university hospital in Japan. Airflow obstruction was defined as a forced expiratory volume in 1 second (FEV₁)/forced vital capacity < 0.7 or as the lower limit of the normal.</p><p>Results</p><p>Among 323 patients presenting with FEV₁/forced vital capacity < 0.7, 43 had asthma and 280 did not. Among the non-asthmatic patients with airflow obstruction, 94 (34%) were non-smokers. A larger number of women than men with airflow obstruction had asthma (26% vs. 7.6%, p < 0.001), or were non-smokers among non-asthmatics (72% vs. 20%, p < 0.001). Non-asthmatic non-smokers, rather than non-asthmatic smokers, asthmatic non-smokers, and asthmatic smokers, exhibited better pulmonary function (median FEV₁: 79% of predicted FEV₁ vs. 73%, 69%, and 66%, respectively, p = 0.005) and less dyspnea on exertion (1% vs. 12%, 12%, and 28%, respectively, p = 0.001). Pulmonary emphysema on thoracic computed tomography was less common in non-smokers (p < 0.001). Using the lower limit of the normal to define airflow obstruction yielded similar results.</p><p>Conclusions</p><p>There are a substantial number of non-smokers with airflow obstruction compatible with COPD in Japan. In this study, airflow obstruction in non-smokers was more common in women and likelier to result in mild functional and pathological abnormalities than in smokers. Further studies are warranted to investigate the long-term prognosis and appropriate management of this population in developed countries, especially in women.</p></div