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The most common pathogenesis for familial Alzheimer's disease (FAD) involves misprocessing/alternative processing of the amyloid precursor protein (APP) by γ-secretase (for review, see Gandy1 and Small and Gandy2). This misprocessing/alternative processing leads to a relative increase in the ratio of the level of a minor γ-secretase reaction product, amyloid-β42 (Aβ42), to that of the major reaction product, amyloid-β40 (Aβ40; Borchelt and colleagues3). Until now, little has been known about γ-secretase function in sporadic AD (SAD). We approached this issue by studying the metabolite peptides, p3-Alcα, that are derived from the processing by α-secretases and γ-secretases of the alcadeinα (Alcα), member of the alcadein (Alc) protein family. Alc proteins colocalize with APP in healthy mouse and SAD human brain,4 but they are entirely distinct from APP in their polypeptide sequence. In neurons, Alc proteins are complexed to APP via X11L adaptor molecules, raising the possibility that Alcs might be sorted and processed together with APP. Experimental evidence supports this reasoning. In the absence of X11L, both Alc and APP proteins are rapidly metabolized by proteolysis.5 Levels of the endogenous APP metabolite, Aβ, are elevated in the brains of X11L-deficient mice.6,7 Thus, taken together with similarities in their structure and cellular distribution, APP and Alc proteins would be predicted to undergo parallel metabolic fates (for APP and X11L, see Gandy1 and Suzuki and Nakaya8; for Alc, see Araki and colleagues,4,5,9). Alc proteins exist in mammalian neurons as 4 isoforms4: Alcα1, Alcα2, Alcβ, and Alcγ. Alcα, Alcβ, and Alcγ are encoded by independent genes, while Alcα1 and Alcα2 are splice variants derived from the Alcα gene. All 3 members of the Alc family (Alcα, Alcβ, and Alcγ) are cleaved by ADAM 10 and ADAM 17, which have been identified as the α-secretases for APP.10–13 Subsequent cleavage of the remaining Alc C-terminal fragments involves predominantly the presenilin 1-(PS1)-dependent γ-secretase, and this reaction liberates a short peptide that we have designated p3-Alcα into cell-conditioned media and into cerebrospinal fluid (CSF). The amino acid sequences of the various alternatively cleaved p3-Alcα peptides in human CSF are shown with those of APP-p3 and Aβ (Fig 1). The current study is based on the hypothesis that examination of AD-related processing of p3-Alcα might reveal evidence for γ-secretase dysfunction in SAD. In so doing, we seek to confirm and extend the report of Yanagida and colleagues,14 who described similar alternative processing of another γ-secretase substrate, APLP. Taken together with the data from Yanagida and colleagues,14 we suggest that multiple γ-secretase substrates are subjected to altered processing in SAD and that this potentially implicates an “acquired” γ-secretase dysfunction that might contribute the pathogenesis of SAD. Figure 1 Amino acid sequences and cleavage sites of p3-Alcα and Aβ in human CSF. The amino acid sequences of p3-Alcα (black-underline) along with the sequences of p3 (gray double-underline) and Aβ40 (black double-underline) of APP. ...

Niemann-Pick disease type C (NP-C) is an autosomal recessive neurovisceral lipid storage disorder biochemically characterized by a defect in intracellular transport of low-density lipoprotein (LDL)-derived cholesterol from the lysosome to other cellular sites. We have found substantial accumulation of GM2 ganglioside in NP-C fibroblasts. The intracellular distribution of GM2 ganglioside was similar to that of cholesterol detected by filipin staining, indicating that the accumulation of GM2 ganglioside is mainly lysosomal. The incorporation of N-acetyl-D-[3H]mannosamine into gangliosides was also increased in NP-C fibroblasts, especially into the GM2 and GM3 fractions. A culture condition which eliminates cholesterol accumulation does not eliminate GM2 accumulation. It is suggested that the accumulation of GM2 ganglioside together with the accumulation of cholesterol is a unique abnormality in NP-C fibroblasts and that the defect in NP-C may involve intracellular transport of both cholesterol and GM2 ganglioside.

A strong-base-induced intramolecular cycloaddition reaction of homophthalic anhydrides has been examined as a method for preparing nonlinear polycyclic peri-hydroxy aromatic compounds

Alcadeins (Alcs) constitute a family of neuronal type I membrane proteins, designated Alc(alpha), Alc(beta), and Alc(gamma). The Alcs express in neurons dominantly and largely colocalize with the Alzheimer amyloid precursor protein (APP) in the brain. Alcs and APP show an identical function as a cargo receptor of kinesin-1. Moreover, proteolytic processing of Alc proteins appears highly similar to that of APP. We found that APP alpha-secretases ADAM 10 and ADAM 17 primarily cleave Alc proteins and trigger the subsequent secondary intramembranous cleavage of Alc C-terminal fragments by a presenilin-dependent gamma-secretase complex, thereby generating "APP p3-like" and non-aggregative Alc peptides (p3-Alcs). We determined the complete amino acid sequence of p3-Alc(alpha), p3-Alc(beta), and p3-Alc(gamma), whose major species comprise 35, 37, and 31 amino acids, respectively, in human cerebrospinal fluid. We demonstrate here that variant p3-Alc C termini are modulated by FAD-linked presenilin 1 mutations increasing minor beta-amyloid species Abeta42, and these mutations alter the level of minor p3-Alc species. However, the magnitudes of C-terminal alteration of p3-Alc(alpha), p3-Alc(beta), and p3-Alc(gamma) were not equivalent, suggesting that one type of gamma-secretase dysfunction does not appear in the phenotype equivalently in the cleavage of type I membrane proteins. Because these C-terminal alterations are detectable in human cerebrospinal fluid, the use of a substrate panel, including Alcs and APP, may be effective to detect gamma-secretase dysfunction in the prepathogenic state of Alzheimer disease subjects.

A strong-base-induced intramolecular cycloaddition reaction of homophthalic anhydrides has been examined as a method for preparing nonlinear polycyclic peri-hydroxy aromatic compounds

Background/Aims: Alzheimer’s disease (AD) is a well-known type of dementia. However, it remains difficult to identify AD in the early stage and to distinguish it from other dementing disorders. We examined glycoproteins in cerebrospinal fluid (CSF) as potential biological markers of AD. Methods: CSF samples were collected from AD, other dementia and nondemented patients. Glycoproteins in CSF were detected by lectin blotting using wheat germ agglutinin (WGA), and sugar chain analysis was performed by isoelectric focusing. Results: In Alzheimer’s CSF, several glycoproteins had lower WGA-binding activities, one of which was sufficiently sensitive and specific to distinguish AD from nondemented controls and other dementias. Further analysis identified this glycosylated protein as transferrin, and altered sugar chain composition of transferrin isoforms was observed despite normal protein levels in CSF. Conclusion: The decreased WGA-binding activity of transferrin in AD is probably due to altered glycosylation of transferrin molecules. Transferrin glycosylation is thus a potential biological marker for AD diagnosis, and changes in this glycosylation may play an important role in the pathophysiology of AD.

Cultured fibroblasts from patients with Niemann-Pick disease type C (NP-C) are characterized by lysosomal accumulation of unesterified cholesterol and a defect in intracellular trafficking of cholesterol. We have found the accumulation of GM2 ganglioside in NP-C fibroblasts [Yano T, Taniguchi M, Akaboshi S, Vanier MT, Tai T, Sakuraba H, et al. Proc Japan Acad 1996;72B:214–219]. In this communication we show that several inhibitors known to inhibit intracellular cholesterol transport, progesterone, imipramine and KN-62, elicit accumulation of not only unesterified cholesterol but also GM2 ganglioside. This finding suggests that intracellular transport of cholesterol may be coupled with that of GM2 ganglioside. The accumulation of free cholesterol and GM2 ganglioside may be a clue for understanding the basic defect of NP-C. Recently NPC1 gene is found by the positional cloning. The mechanism of accumulating of GM2 ganglioside should be further investigated by studying of the functions of NPC1 gene.

Niemann–Pick disease type C (NP-C) is an autosomal recessive disorder characterized by the sequestration and trapping of endocytosed cholesterol in lysosomes. The NPC1 gene on chromosome 18 was recently identified but its physiological function remains unknown. We have studied the lipid compositions of cultured human NP-C fibroblasts and mouse SPM-3T3 cell line derived from the C57BL/KsJ NP-C model mouse, which belongs to the same complementation group. Fibroblasts derived from apparently normal age-matched individuals and a subline of SPM-3T3 cells which restores cholesterol metabolism by transfer of human chromosome 18 were used as controls. Levels of free cholesterol in whole cell homogenates increased about 1.5-fold in human NP-C fibroblasts and mouse SPM-3T3 cells, while in the plasma membrane, cholesterol content did not significantly change in NP-C fibroblasts but rather decreased in SPM-3T3 cells. The total phospholipid content did not significantly change; however, among phospholipid head groups, increases in sphingomyelin and decreases in other classes were observed in human NP-C fibroblasts and mouse SPM-3T3 cells. The ratios of saturated fatty acids to unsaturated fatty acids increased in both human and mouse cells. The increase was also confirmed in the plasma membrane fraction of SPM-3T3 cells. Membrane fluidity was examined using a 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescent probe. The DPH anisotropy values were markedly increased in NP-C fibroblasts and in SPM-3T3 cells. The results suggest that a NP-C mutation causes complex alterations in cellular lipid contents and biophysical properties of the membrane.

A strong base-induced intramolecular cycloaddition reaction homophthalic anhydride has been examined as a method for preparing polycyclic perihydroxy aromatic compounds.

A strong base-induced intramolecular cycloaddition reaction homophthalic anhydride has been examined as a method for preparing polycyclic perihydroxy aromatic compounds.

Background Alcadeinα (Alcα) is a neuronal membrane protein that colocalizes with the Alzheimer's amyloid-β precursor protein (APP). Successive cleavage of APP by β- and γ-secretases generates the aggregatable amyloid-β peptide (Aβ), while cleavage of APP or Alcα by α- and γ-secretases generates non-aggregatable p3 or p3-Alcα peptides. Aβ and p3-Alcα can be recovered from human cerebrospinal fluid (CSF). We have previously reported alternative processing of APP and Alcα in the CSF of some patients with sporadic mild cognitive impairment (MCI) and AD (SAD). Results Using the sandwich enzyme-linked immunosorbent assay (ELISA) system that detects total p3-Alcα, we determined levels of total p3-Alcα in CSF from subjects in one of four diagnostic categories (elderly controls, MCI, SAD, or other neurological disease) derived from three independent cohorts. Levels of Aβ40 correlated with levels of total p3-Alcα in all cohorts. Conclusions We confirm that Aβ40 is the most abundant Aβ species, and we propose a model in which CSF p3-Alcα can serve as a either (1) a nonaggregatable surrogate marker for γ-secretase activity; (2) as a marker for clearance of transmembrane domain peptides derived from integral protein catabolism; or (3) both. We propose the specification of an MCI/SAD endophenotype characterized by co-elevation of levels of both CSF p3-Alcα and Aβ40, and we propose that subjects in this category might be especially responsive to therapeutics aimed at modulation of γ-secretase function and/or transmembrane domain peptide clearance. These peptides may also be used to monitor the efficacy of therapeutics that target these steps in Aβ metabolis