Nobuo Ida

Cerebrospinal fluid Aβ42 is increased early in sporadic Alzheimer's disease and declines with disease progression

All mutations known to cause familial Alzheimers disease (AD) act by increasing the levels of soluble β‐amyloid peptide (Aβ), especially the longer form, Aβ42. However, in vivo elevation of soluble Aβ in sporadic AD has so far not been shown. In the present study, we used enzyme‐linked immunosorbent assays specific for Aβ42 and Aβ40 to investigate cerebrospinal fluid from sporadic AD at different stages of disease severity, to clarify the roles of Aβ42 and Aβ40 during disease progression. We also evaluated three other groups—one group of patients with mild cognitive impairment who were at risk of developing dementia, a cognitively intact, nondemented reference group diagnosed with depression, and a perfectly healthy control group. We found that Aβ42 is strongly elevated in early and mid stages of AD, and thereafter it declines with disease progression. On the contrary, Aβ40 levels were decreased in early and mid stages of AD. The group of cognitively impaired patients and the depression reference group had significantly higher levels of Aβ42 than the healthy control group, implying that Aβ42 is increased not only in AD, but in other central nervous system conditions as well. Our data also point out the importance of having thoroughly examined control material. The initial increase and subsequent decrease of Aβ42 adds a new biochemical tool to follow the progression of AD and might be important in the monitoring of therapeutics. Ann Neurol 1999;45:504–511

The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein.

We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild‐type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild‐type APP and mutations implicated in Alzheimers disease and another brain beta‐amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane‐inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11–15 in the carbohydrate domain, the other encoded by exons 16–17 in the juxtamembraneous beta‐amyloid domain. Treatment of the cells with the N‐glycosylation inhibitor tunicamycin induced missorting of wild‐type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta‐amyloid‐dependent membrane proximal signal was the major contributor to axonal sorting, while N‐glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS‐insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta‐amyloid domain plays a major role in axonal delivery of APP.

Regulation of Amyloid Precursor Protein Catabolism Involves the Mitogen-Activated Protein Kinase Signal Transduction Pathway

Catabolic processing of the amyloid precursor protein (APP) is subject to regulatory control by protein kinases. We hypothesized that this regulation involves sequential activation of the enzymes mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated protein kinase (ERK). In the present investigation, we provide evidence that MEK is critically involved in regulating APP processing by both nerve growth factor and phorbol esters. Western blot analysis of the soluble N-terminal APP derivative APPsdemonstrated that the synthetic MEK inhibitor PD 98059 antagonized nerve growth factor stimulation of both APPs production and ERK activation in PC12 cells. Moreover, PD 98059 inhibited phorbol ester stimulation of APPs production and activation of ERK in both human embryonic kidney cells and cortical neurons. Furthermore, overexpression of a kinase-inactive MEK mutant inhibited phorbol ester stimulation of APP secretion and activation of ERK in human embryonic kidney cell lines. Most important, PD 98059 antagonized phorbol ester-mediated inhibition of Aβ secretion from cells overexpressing human APP695 carrying the “Swedish mutation.” Taken together, these data indicate that MEK and ERK may be critically involved in protein kinase C and nerve growth factor regulation of APP processing. The mitogen-activated protein kinase cascade may provide a novel target for altering catabolic processing of APP.

Cerebral changes and cerebrospinal fluid beta-amyloid in Alzheimer's disease : a study with quantitative magnetic resonance imaging

Pathological and biochemical studies indicate that β-amyloid (βA4) deposition is a hallmark in the pathogenesis of Alzheimers disease (AD).1–4 Neuroimaging studies demonstrate that the respective cerebral changes primarily strike the temporal lobe and the amygdala-hippocampus complex and may be reliably assessed using quantitative magnetic resonance imaging (MRI).5,6 Therefore one may expect that reduced βA4-levels are significantly correlated with measures of the temporal lobe rather than global cerebral atrophy in AD patients. To test this hypothesis in a clinical study, cerebrospinal fluid concentrations of total β A4 and its major C-terminal variations β A4 1–40 and β A4 1–42 were compared with cerebral changes as assessed by quantitative magnetic resonance imaging (MRI). Significantly (P < 0.05) reduced β A4 1–40 and β A4 1–42 levels were found in the AD patients (17 female; six male; AD/NINCDS-ADRDA-criteria)7 in comparison to the patients with major depression (seven female; two male; DSM-III-R).8 Within the AD group, βA4 and β xA4 1–42 levels were significantly correlated with the volume of the temporal lobes (r = 0.46 and r = 0.48, respectively) but none of the other volumetric measures. These findings indicate that changes in cerebral β A4 levels contribute to temporal lobe atrophy in AD and support the possibility that βA4 is central to the etiology of AD.

Rapid cellular uptake of Alzheimer amyloid βA4 peptide by cultured human neuroblastoma cells

Cerebral deposition of βA4 (β‐amyloid) peptide is a major pathological feature of Alzheimers disease. Although the mechanism of βA4 production from cells has been investigated extensively, so far little is known about the catabolism of the peptide. We report here that the human neuroblastoma cell line SH‐SY5Y can rapidly clear βA4 in the culture medium. The clearance was not due to the degradation by extracellularly released protease, but rather due to intracellular degradation after cellular uptake. This clearance activity was specific to SH‐SY5Y cells among several cell types examined. Some of the βA4‐derived peptides lacking the N‐terminal part of the molecule were not catabolized, suggesting a specific interaction between the cells and βA4. Although most of the peptide was degraded after uptake, small amounts of peptide was accumulated in insoluble fractions of the cells and remained stable for several days. These observations suggest that this uptake‐degradation activity may contribute to AD pathogenesis in two different ways: either to prevent the amyloid deposition by reducing extracellular βA4 concentrations, or to promote the deposition by producing insoluble seeds for amyloid formation.

Analysis of presenilin 1 and presenilin 2 expression and processing by newly developed monoclonal antibodies.

Because distinct mutations in presenilin 1 and presenilin 2 are a major cause of early‐onset familial Alzheimers disease, we generated four monoclonal antibodies for the identification, localization, and investigation of presenilins in various cell lines and tissues from patients and controls. We show that these antibodies are specific for the N‐ and C‐terminal domains of human presenilin 1 and presenilin 2. They recognize presenilin full‐length proteins and their ≈28–35 kDa N‐terminal fragments and ≈18–20 kDa C‐terminal fragments. None of the antibodies showed cross‐reaction in their specific detection ability. We demonstrated that presenilin 1 and presenilin 2 are proteolytically processed in human glioma cell lines, transfected and untransfected human neuroblastoma SH‐SY5Y cells, COS‐7 cells, rat cerebellar neuronal ST15 cells, mouse and human brain. Remarkably, we observed that presenilin 2 is alternatively cleaved during apoptosis, producing smaller C‐terminal fragments. By analyzing the subcellular distribution of presenilins, we found reticular and fine vesicular staining throughout the cell bodies. In addition, staining of Golgi compartments and the perinuclear envelope was observed. Alzheimers disease brain showed strong immunoreactivity of presenilin 1 in reactive astrocytes and senile plaques. This high expression of presenilin 1 may explain the increased production and accumulation of the amyloid‐β peptide in patients with sporadic Alzheimers disease in the absence of familial presenilin mutation. J. Neurosci. Res. 56:405–419, 1999.

Novel Effects of FCCP [Carbonyl Cyanide p‐(Trifluoromethoxy)phenylhydrazone] on Amyloid Precursor Protein Processing

Abstract: Amyloidogenic processing of the β‐amyloid precursor protein (APP) has been implicated in the pathology of Alzheimer’s disease. Because it has been suggested that catabolic processing of the APP holoprotein occurs in acidic intracellular compartments, we studied the effects of the protonophore carbonyl cyanide p‐(trifluoromethoxy)phenylhydrazone (FCCP) and the H+‐ATPase inhibitor bafilomycin A1 on APP catabolism in human embryonic kidney 293 cells expressing either wild‐type or “Swedish” mutant APP. Unlike bafilomycin A1, which inhibits β‐amyloid production in cells expressing mutant but not wild‐type APP, FCCP inhibited β‐amyloid production in both cell types. Moreover, the effects of FCCP were independent of alterations in total cellular APP levels or APP maturation, and the concentrations used did not alter either cellular ATP levels or cell viability. Bafilomycin A1, which had no effect on β‐amyloid production in wild‐type cells, inhibited endocytosis of fluorescent transferrin, whereas concentrations of FCCP that inhibited β‐amyloid production in these cells had no effect on endosomal function. Thus, in wild‐type‐expressing cells it appears that the β‐amyloid peptide is not produced in the classically defined endosome. Although bafilomycin A1 decreased β‐amyloid release from cells expressing mutant APP but not wild‐type APP, it altered lysosomal function in both cell types, suggesting that in normal cells β‐amyloid is not produced in the lysosome. Although inhibition of β‐amyloid production by bafilomycin A1 in mutant cells may occur via changes in endosomal/lysosomal pH, our data suggest that FCCP inhibits wild‐type β‐amyloid production by acting on a bafilomycin A1‐insensitive acidic compartment that is distinct from either the endosome or the lysosome.