Ursula Mönning

Regulation and Expression of the Alzheimer's β/A4 Amyloid Protein Precursor in Health, Disease, and Down's Syndromea

A four‐ to fivefold overexpression of the gene for the Alzheimer β/A4 amyloid precursor protein (APP) in individuals with Downs Syndrome (DS) appears to be responsible for the fifty year earlier onset of Alzheimers disease (AD) pathology in DS compared to the normal population. It is therefore likely that a deregulated overexpression of the APP gene is a risk factor for the β/A4 amyloid formation. To test this hypothesis and to get a better understanding of how APP expression is regulated, we studied the 5′ control region of the human APP gene, alternative splicing of the 19 APP exons, and APP biogenesis, metabolism and function. The analysis of the APP promoter revealed its similarity with those of housekeeping genes by the presence of a GC‐rich region around the transcription start site and the lack of a TATA box. Gene transfer experiments showed this GC‐rich region to contain overlapping binding sites for different transcription factors whose binding is mutually excluded. An imbalance between these factors may cause APP overexpression and predispose to AD pathology. Another putative risk factor for AD is regulation of splicing of exon 7 in APP mRNAs which changes in brain during aging. This is relevant for APP processing since exon 7 codes for a Kunitz protease inhibitory domain. Investigation of further splicing adjacent to the β/A4 exons 16 and 17 which might also interfere with APP processing led to the identification of the leukocyte‐derived (L‐APP) splice forms which lack exon 15. In brain this splicing occurs in activated astrocytes and microglia. The localization of APP at synaptic sites in brain suggests that APP regulation and expression are critical determinants of a potential and early impairment of central synapses. This may be the case during pathological evolution of AD and DS when β/A4 derived from synaptic APP is converted to β/A4 amyloid by radical generation.

Cerebrospinal Fluid Levels of Amyloid Precursor Protein and Amyloid β-Peptide in Alzheimer’s Disease and Major Depression – Inverse Correlation with Dementia Severity

Alzheimer’s disease (AD) is the most common neurodegenerative disorder characterized by progressive dementia that ultimately leads to death. Histopathological hallmarks of AD include brain amyloid deposits and neurofibrillary tangles. Major depression is a frequent diagnosis in every gerontopsychiatric clinic that sees patients with both cognitive and affective disorders. Many depressed patients, in fact, are clinically characterized by cognitive impairments. Thus, an assay that excludes – or confirms – probable AD in cognitively impaired patients is desirable. Such assays may use protein markers that are derived from such histopathologically relevant molecules as the amyloid precursor protein (APP) and its derivatives including the amyloid β-peptides (Aβ). To evaluate the differential diagnostic properties of cerebrospinal fluid (CSF) Aβ and secreted soluble ectodomain (APPs), we quantitated CSF levels of these measures in AD patients and compared them to age-matched control patients with major depression. CSF levels of APPs and Aβ were similar in patients with AD or major depression, and the apolipoprotein E genotype had no influence on CSF levels of Aβ in AD patients. Measurement of Aβ peptide using a novel zinc/copper capture ELISA that detects aggregated Aβ peptides as well demonstrated similar levels in AD and major depression. In AD patients, CSF levels of total Aβ (Aβ1–40 plus Aβ1–42) were inversely correlated with a functional measure of dementia severity (NOSGER), suggesting that CSF levels of Aβ decrease with advancing severity of AD. Thus, CSF levels of Aβ are not useful for the differentiation of AD from major depression. However, CSF levels of Aβ reflect the severity of dementia and may be useful as biological markers of the stage of the disease.

Amyloid precursor protein secretion and βA4 amyloid generation are not mutually exclusive

The cellular factors regulating the generation of βA4 from the amyloid precursor protein (APP) are unknown. Protein phosphorylation by protein kinase C (PKC) has been found to influence the processing and metabolism of APP. In this report, we show that in the human neuroblastoma cell line SY5Y, βA4 generation from full‐length APP is not changed by PKC activation whereas production of the non‐amyloidogenic secretory fragment (APPsec) and of the C‐terminal fragment of βA4 (p3) are stimulated. In addition, βA4 generation from the membrane inserted C‐terminal 100 residues (SPA4CT) of APP is stimulated by PKC activation. Accordingly attempts to divert APP processing from the amyloidogenic, βA4‐generating, to the non‐amyloidogenic, secretory, pathway, have to address the nature and regulation of the two pathways and/or of the process leading to the cleavage of APP at the C‐terminus of the βA4 domain. The data reported here suggest that these mechanisms are cell‐type specific.

Quantitative changes in the amyloid βA4 precursor protein in Alzheimer cerebrospinal fluid

The major three secretory isoforms of Alzheimer beta A4 amyloid precursor protein (APP) were quantified in cerebrospinal fluid (CSF) using (1) a newly developed enzyme-linked immunosorbent assay (ELISA) and (2) densitometric analysis of CSF Western blots. The protease inhibitor-containing APP751/770 isoforms represented an average of 10.5% of total APP in CSF of patients with Alzheimers disease (AD, n = 22), multi-infarct dementia (MID, n = 5) and normal controls (n = 10). APP levels in CSF did not depend on total CSF protein. Both findings are inconsistent with a hematogeneous origin of APP in CSF and suggest an intracerebral source. Total APP, APP695 and APP751/770 were significantly decreased in the AD and in the MID groups, but were not correlated to the ages of patients or controls.

Synthesis and secretion of Alzheimer amyloid βA4 precursor protein by stimulated human peripheral blood leucocytes

Alzheimer amyloid precursor proteins (APP) are actively secreted by stimulated human peripheral mononuclear blood leucocytes (PMBLs). Induction of APP transcription, translation and secretion was observed with several T cell mitogens but was highest with phytohemagglutinin. The time course of induction is similar to that reported for IL‐2 and IL‐2 receptor. We suggest that APP may play an important role in the construction of the immunological network and the differentiation of T cells.

Transforming growth factor β mediates increase of mature transmembrane amyloid precursor protein in microglial cells

By using the immortalized microglial cell line BV‐2, we show that the high expression of theβA4 amyloid precursor protein (APP), its biogenesis and metabolism is modulated by TGF β, a cytokine with immunosuppressive activity, and by the microglia‐stimulating agent LPS. TGFβ induces accumulation of cellular mature APP, the putative precursor of the amyloid subunit of Alzheimers disease. LPS leads to an increase in cellular immature, non‐amyloidogenic APP and secretion of also non‐amyloidogenic APP fragments. We also demonstrate a functional involvement of ECM molecules in the regulation of microglial APP expression at mRNA and protein level by TGFβ and LPS.

Apolipoprotein E-4 gene dose in clinically diagnosed Alzheimer's disease: prevalence, plasma cholesterol levels and cerebrovascular change

SummaryThe prevalence of the apolipoprotein E-4 allele (ApoE-4) was significantly higher in a referral population of 40 patients with clinically diagnosed Alzheimers disease than in a sample of non-demented elderly controls (P<0.01). The highest plasma cholesterol levels were found in demented patients homozygotic for Apo E-4, but no significant increases of glucose, triglycerides and thyroxine or of leuko-araiosis and brain infarcts were verified in this preliminary study.

In‐vitro matured human macrophages express Alzheimer's βA4‐amyloid precursor protein indicating synthesis in microglial cells

Microglia which are consistently associated with Alzheimers disease (AD) senile plaques are part of the mononuclear phagocyte system. In‐vitro matured human monocyte‐derived macrophages feature many immunological characteristics of microglia. We found strong constitutive expression of Alheimers βA4‐amyloid precursor protein (APP) in human mononuclear phagocytes after terminal in‐vitro maturation from monocytes to macrophages. Amyloid has previously been found to be associated with microglia in AD brains, however, it remained unclear whether the material was synthesized in or had been phagocytosed by the cells. The findings presented here support the assumption that brain microglia may contribute to APP synthesis in AD brain.

Expression of the APP Gene Family in Brain Cells, Brain Development and Aging

The Alzheimers beta A4-amyloid protein precursor (APP) and the APP-like proteins (APLPs) are transmembrane glycoproteins with a similar modular domain structure. Alternatively spliced exons found in both genes comprise a Kunitz protease inhibitor domain encoding exon, and another exon within the divergent regions adjacent to the transmembrane domain, i.e. exon 15 of the APP gene and an exon encoding 12 residues in APLP2. Omission of the latter exons in L-APP and L-APLP2 isoforms, respectively, generates a functional recognition sequence for xylosyltransferase-mediated addition of glycosaminoglycans and proteoglycan formation. In this paper, we summarize our analyses of the regulated expression of these alternatively spliced exons in APP and APLP2 in primary cultured rat brain cells, rat brain development and aging. In conjunction with additional data for the human brain, these data provide important clues for understanding the functional significance of alternative splicing and glycosylation in APP biology. On the basis of recent results showing a higher amyloidogenicity of exon 15 encoding APP than L-APP isoforms, we further discuss the potential significance of the low levels of L-APP in neurons for the susceptibility of the brain towards Alzheimers disease.

Regulation of APP Expression, Biogenesis and Metabolism by Extracellular Matrix and Cytokinesa

We have identified and characterized the ligand binding properties of the Alzheimers disease (AD) βA4 amyloid protein precursor (APP), mapped the APP ligand binding sites and analyzed the regulation of APP expression, biogenesis and metabolism by components of the extracellular matrix (ECM) and cytokines.