Andreas Weidemann

Identification, biogenesis, and localization of precursors of Alzheimer's disease A4 amyloid protein

To study the putative precursor proteins (PreA4(695), PreA4(751), and PreA4(770] of Alzheimers disease A4 amyloid protein, polyclonal and monoclonal antibodies were raised against a recombinant bacterial PreA4(695) fusion protein. These antibodies were used to identify the precursors in different cell lines as well as in human brain homogenates and cerebrospinal fluid (CSF). The precursors are tyrosine-sulfated, O- and N-glycosylated membrane proteins and have half-lives of 20-30 min in cells. Cells express the polypeptides at their surface but also secrete C-terminal truncated proteins into the medium. These proteins are also found in CSF of both Alzheimers disease patients and normal individuals. The proteins are derived from their cognate membrane-associated forms by proteolysis and have apparently lost the cytoplasmic and the transmembrane domains. Since the latter contributes to the A4 amyloid sequence, it seems possible that this proteolytic cleavage represents the first step in the formation of A4 amyloid deposits.

Identification, transmembrane orientation and biogenesis of the amyloid A4 precursor of Alzheimer's disease.

The precursor of the Alzheimers disease‐specific amyloid A4 protein is an integral, glycosylated membrane protein which spans the bilayer once. The carboxy‐terminal domain of 47 residues was located at the cytoplasmic site of the membrane. The three domains following the transient signal sequence of 17 residues face the opposite side of the membrane. The C‐terminal 100 residues of the precursor comprising the amyloid A4 part and the cytoplasmic domain have a high tendency to aggregate, and proteinase K treatment results in peptides of the size of amyloid A4. This finding suggests that there is a precursor‐product relationship between precursor and amyloid A4 and we conclude that besides proteolytic cleavage other events such as post‐translational modification and membrane injury are primary events that precede the release of the small aggregating amyloid A4 subunit.

Localization of Alzheimer βA4 amyloid precursor protein at central and peripheral synaptic sites

We have recently shown that the amyloid beta A4 precursor protein (APP) is synthesized in neurons and undergoes fast axonal transport to synaptic sites [Koo et al., Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 1561-1565]. Using immunofluorescence, laser confocal microscopy and immunoelectron microscopy with simultaneous detection of APP and synaptophysin, we now report a preferential localization of APP at synaptic sites of human and rat brain and at neuromuscular junctions. APP is further found on vesicular elements of neuronal perikarya, dendrites and axons. The synaptic localization of APP implies (1) a role of APP in physiological synaptic activity and (2) a potential and early impairment of central synapses when synaptic APP is converted to beta A4 amyloid during the pathological evolution of Alzheimers disease and Downs syndrome.

Proteolytic Processing of the Alzheimer’s Disease Amyloid Precursor Protein within Its Cytoplasmic Domain by Caspase-like Proteases

Alzheimer’s disease is characterized by neurodegeneration and deposition of βA4, a peptide that is proteolytically released from the amyloid precursor protein (APP). Missense mutations in the genes coding for APP and for the polytopic membrane proteins presenilin (PS) 1 and PS2 have been linked to familial forms of early-onset Alzheimer’s disease. Overexpression of presenilins, especially that of PS2, induces increased susceptibility for apoptosis that is even more pronounced in cells expressing presenilin mutants. Additionally, presenilins themselves are targets for activated caspases in apoptotic cells. When we analyzed APP in COS-7 cells overexpressing PS2, we observed proteolytic processing close to the APP carboxyl terminus. Proteolytic conversion was increased in the presence of PS2-I, which encodes one of the known PS2 pathogenic mutations. The same proteolytic processing occurred in cells treated with chemical inducers of apoptosis, suggesting a participation of activated caspases in the carboxyl-terminal truncation of APP. This was confirmed by showing that specific caspase inhibitors blocked the apoptotic conversion of APP. Sequence analysis of the APP cytosolic domain revealed a consensus motif for group III caspases ((IVL)ExD). Mutation of the corresponding Asp664 residue abolished cleavage, thereby identifying APP as a target molecule for caspase-like proteases in the pathways of programmed cellular death.

Biogenesis and metabolism of Alzheimer’s disease Aβ amyloid peptides

Biochemical and genetic evidence indicates the balance of biogenesis/clearance of Abeta amyloid peptides is altered in Alzheimers disease. Abeta is derived, by two sequential cleavages, from the receptor-like amyloid precursor protein (APP). The proteases involved are beta-secretase, identified as the novel aspartyl protease BACE, and gamma-secretase, a multimeric complex containing the presenilins (PS). Gamma-secretase can release either Abeta40 or the more aggregating and cytotoxic Abeta42. Secreted Abeta peptides become either degraded by the metalloproteases insulin-degrading enzyme (IDE) and neprilysin or metabolized through receptor uptake mediated by apolipoprotein E. Therapeutic approaches based on secretase inhibition or amyloid clearance are currently under development.

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.

Alpha 2-macroglobulin synthesis in interleukin-6-stimulated human neuronal (SH-SY5Y neuroblastoma) cells Potential significance for the processing of Alzheimer β-amyloid precursor protein

Cultured human neuronal (SH‐SY5Y neuroblastoma) cells synthesize and secrete the potent protease inhibitor alpha 2‐macroglobulin (a2M) upon stimulation with interleukin‐6(IL‐6) indicating that alpha 2‐macroglobulin behaves as an acute‐phase protein in the human central nervous system. Exogenous addition of a2M to the cultured neuronal cells resulted in only a slight inhibition of Alzheimer βA4‐amyloid precursor protein (APP) synthesis, but markedly inhibited its secretion pointing to the possibility that a2M may affect the proteolytic APP processing. Evidence is provided that IL‐6 and a2M are involved in Alzheimers disease pathogenesis.

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.

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.

Alternative transcripts of presenilin-1 associated with frontotemporal dementia.

&NA; We have analyzed the expression of Alzheimers disease‐associated presenilin 1 (PS1) in various neurodegenerative disorders. Western blotting identified PS1 N‐ and C‐terminal fragments similarly in the cortex of controls, Parkinson, Huntington and schizophrenia subjects. Additional PS1 immunoreactive species of 42 and 46 kDa were present in six out of seven cases of sporadic frontotemporal dementia (FTD) and these were particularly prominent in two cases. RT‐PCR analysis using nested primers showed the presence of PS1 gene products with deletions within the exon 4–8 region. Our results suggest that alternative transcription of PS1 may be associated with FTD.