Kikuko Tanaka

Proteolytic processing of presenilin-1 (PS-1) is not associated with Alzheimer's disease with or without PS-1 mutations.

Cerebral presenilin‐1 protein (PS‐1) is normally composed of the amino‐terminal fragment (NTF) with M r 28 kDa and the carboxy‐terminal fragment (CTF) with 18 kDa. We analyzed human PS‐1 in brains with early‐onset familial Alzheimers disease (FAD) with and without PS‐1 mutations to study whether mutated PS‐1 was abnormally metabolized. Cerebral PS‐1 were found to be cleaved into two fragments of NTF and CTF independently of the occurrence of PS‐1 mutation in human brains. A small portion of PS‐1 was recently found to suffer another processing by caspase‐3, an apoptosis‐related cysteine protease. In contrast to the recent finding that the Volga‐German mutation on presenilin‐2 (PS‐2) affects the increasing caspase‐3 PS‐2 fragment, the PS‐1 mutation did not cause a significant change in PS‐1 fragmentation. We conclude that PS‐1 fragmentation and other (probably caspase‐3‐mediated) digestion following apoptosis occur independently of PS‐1 mutations.

Serum transthyretin monomer in patients with familial amyloid polyneuropathy.

Although dissociation of the transthyretin (TTR) tetramer is suspected of being the first step in amyloid fibril formation in hereditary TTR amyloidosis, including familial amyloid polyneuropathy (FAP), the TTR monomer has never been examined in vivo. Therefore, we analyzed the TTR monomer in the serum of FAP patients and normal individuals. Free TTR monomer was detected in both groups using gel filtration chromatography and immunoblotting. Both the mean concentration of free TTR monomer and the total serum TTR were significantly lower in FAP patients than in normal individuals. Moreover, in FAP patients, mass spectrometry showed that the variant TTR monomer was markedly decreased compared with the wild-type TTR monomer. These findings suggest that the free variant TTR monomer is unstable in serum, and that it aggregates in deposits in various organs or is adsorbed by preexisting amyloid fibrils before it is degraded.

Mutant presenilin (A260V) affects Rab8 in PC12D cell

Most familial early-onset Alzheimers disease (FAD) is caused by mutations in the presenilin-1 (PS1) gene. Abeta is derived from amyloid precursor protein (APP) and an increased concentration of Abeta 42 is widely believed to be a pathological hallmark of abnormal PS function. Therefore, the interaction between PS1 and APP is a central theme in attempts to clarify the molecular mechanism of AD. To examine the effect of PS1 mutations on APP metabolism, we made PC12D cell lines that express human PS1 or mutant PS1 (A260V). In PC12D cells expressing the PS1A260V mutant, we found that Rab8, a GTPase involved in transport from the trans-Golgi network (TGN) to the plasma membrane (PM), was significantly reduced in PC12D cells expressing the A260V mutant and that APP C-terminal fragment (CTF), the direct precursor of Abeta, accumulated in the heavy membrane fraction including membrane vesicles involved in TGN-to-PM transport. Furthermore, the total intracellular Abeta production was reduced in these cells. Combined together, we have observed that PS1 mutation disturbs membrane vesicle transport, resulting in prolonged residence of APP CTF during TGN-to-PM transport pathway. Therefore, it is highly likely that reduction of Abeta is closely related to the retention of APP CTF during TGN-to-PM transport.

Secretory cleavage site of Alzheimer amyloid precursor protein is heterogeneous in Down's syndrome brain

Aβ (β/A4) is the major constituent of brain amyloid in Alzheimers disease (AD), Downs syndrome (DS) and normal aged persons. This protein is presumably derived by normal proteolysis from a precursor protein (APP). In this study, C‐terminal fragments of APP in a Tris/Triton soluble fraction were partially purified from DS brain by heparin‐affinity and reverse phase chromatography, and analyzed by N‐terminal amino acid sequencing after SDS polyacrylamide gel electrophoresis and Western blotting. We found at least six different C‐terminal fragments including those with the entire Aβ region. These results suggest that secretory processing of APP is heterogeneous and generates amyloidogenic C‐terminal fragments.

Amyloid β-protein precursor (APP) of cultured cells: Secretory and non-secretory forms of APP

Overproduction or aberrant catabolism of the predicted amyloid beta-protein precursor (APP) is suspected as the cause of amyloid deposition in Alzheimers disease and Downs syndrome brains. For possible in vitro experiments of amyloid formation, we have examined the expression of APP in various cultured cells. We found two types of APP producing cell lines. PC12h (rat pheochromocytoma) and HL-60 (human acute promyelocytic leukemia) cells produce a secretory form that is released into the culture medium, while Bu-17 (human glioma) cells synthesize only a non-secretory form that accumulates at the cell surface. APP immunoreactivity on the latter cells was detected at the tips of cell processes or growth cones. These observations indicate that the nonsecretory form of APP may play a role in cell contact or adhesion.

The immunoreactive profile at the N-terminal region of Aβ1–3/940 but not Aβ 1–42 changes with transition from monomer/dimer to further peptide aggregates

Using site-specific antibodies, we assessed the effect of aggregation of various length forms of A beta on the immunoreactive profile of the peptides. All of the antibodies tested reacted with monomeric/dimeric forms of A beta 1-42 and its further aggregates. However, antibodies directed against the 1-24 region of A beta reacted weakly or not at all with A beta 1-39/40 monomers or dimers, but immunoreactivity was enhanced substantially following peptide incubation and aggregation. These results suggest that the conformation of the N-terminal region of monomeric and dimeric A beta 1-39/40 is different from that of aggregated forms, whereas the longer A beta 1-42 does not significantly change its N-terminal conformation during beta-sheet fibril formation. These immunochemical results are consistent with previous structural data, and help to explain the differential effects of A beta 1-39/40 and 1-42 on fibril formation in brain.

Secretory cleavage of β-amyloid precursor protein in the cerebral white matter produces amyloidogenic carboxyl-terminal fragments

To elucidate the metabolic process generating amyloid-beta protein (A beta) from beta-amyloid precursor protein (APP) in human brain, we partially purified secretory forms and carboxyl-terminal fragments (CTFs) of APP from the white matter of a Downs syndrome brain. We obtained secretory forms of APP which lack the entire A beta sequence and CTFs which contain the full-length A beta from the cerebral white matter. Some A beta-lacking secretory APP isoforms in the white matter were derived from APP695. These results suggest that amyloidogenic CTFs can be produced by secretory cleavage of APP which is anterogradely transported through the axon in human brain.

Presenilin-1 exists in the axoplasm fraction in the brains of aged Down's syndrome subjects and non-demented individuals.

Missense mutations in the presenilin-1 (PS-1) gene are known to be responsible for early-onset familial Alzheimers disease (AD). The normal physiological functions of PS-1 are still incompletely understood, although data on the intracellular localization of PS-1 are accumulating, indicating that it exists mainly in endoplasmic reticulum and Golgi compartments. To investigate the localization and functions of PS-1 in the human brain, we separated axoplasm fractions from the cerebral white matter of Downs syndrome (DS) subjects with AD pathology and non-demented individuals using the axonal flotation method, and analyzed them immunocytochemically. All axoplasm fractions contained the 28-34 kDa amino-terminal fragment and the 18 kDa carboxy-terminal fragment of PS-1, although there was no specific abnormality of this protein in the DS brains with AD pathology. This finding indicates that there is intracellular trafficking of PS-1 through the axons in the human brain, and thus provides new information about the physiology of PS-1.

A monoclonal antibody Hy20-54-16-3L to lambda (λ) light chain of human immunoglobulin reacts with amyloid in Alzheimer's disease brain

We have previously described a monoclonal antibody Hy20-54-16-3L raised against crude primary (AL) amyloid fibrils that preferentially labelled amyloid deposits in the brains of patients with Alzheimers disease (AD) (Ishii et al., Neuropathol. Appl. Neurobiol., 12 (1986) 441-445). In the present study, we analyzed the protein from an AL-amyloid laden spleen by gel filtration and SDS-PAGE; a partial amino acid sequence of the Hy20-54-16-3L-immunoreactive band excised from Western blots was determined and found to be a lambda light chain of human immunoglobulins. Additionally we have confirmed that this monoclonal antibody reacts with amyloid in AD brain by using a peroxidase-linked immunoelectron microscope technique. It is concluded that an epitope shared with a lambda light chain is included in or associated with amyloid deposits in the AD brain.

Secretory form of β-amyloid precursor protein is much abundantly contained in the cerebral white matter in human brain

To elucidate the metabolic process of beta-amyloid precursor proteins (APP) in cerebral gray and white matter of the human brain, we compared the content and characteristics of secretory APP between these two parts. The white matter contained much more secretory APP than the gray matter in both a control and a Downs syndrome brain, but no difference in the characteristics of the APP isoforms was detected. Our results suggest that, in the human brain, a considerable amount of APP is carried by axonal transport, during which some of the APP isoforms are processed to their secretory forms.