Krzysztof Paliga

What the evolution of the amyloid protein precursor supergene family tells us about its function.

The Alzheimers disease amyloid protein precursor (APP) gene is part of a multi-gene super-family from which sixteen homologous amyloid precursor-like proteins (APLP) and APP species homologues have been isolated and characterised. Comparison of exon structure (including the uncharacterised APL-1 gene), construction of phylogenetic trees, and analysis of the protein sequence alignment of known homologues of the APP super-family were performed to reconstruct the evolution of the family and to assess the functional significance of conserved protein sequences between homologues. This analysis supports an adhesion function for all members of the APP super family, with specificity determined by those sequences which are not conserved between APLP lineages, and provides evidence for an increasingly complex APP superfamily during evolution. The analysis also suggests that Drosophila APPL and Caenorhabditis elegans APL-1 may be a fourth APLP lineage indicating that these proteins, while not functional homologues of human APP, are similarly likely to regulate cell adhesion. Furthermore, the betaA4 sequence is highly conserved only in APP orthologues, strongly suggesting this sequence is of significant functional importance in this lineage.

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.

Amyloid precursor-like protein 1 accumulates in neuritic plaques in Alzheimer’s disease

Abstract The Alzheimer’s disease (AD) β-amyloid precursor protein (APP) and the amyloid precursor-like protein 1 (APLP1) and 2 (APLP2) are members of a superfamily of proteins that appear functionally related. Although APLPs are highly homologous to APP in the N- and C-terminal domains, they lack the βA4/amyloid peptide, i.e., the main constituent of neuritic plaques in AD. To assess a potential role of APLP1 in AD, we have determined its immunohistochemical distribution in human hippocampal formation, a structure which is strongly affected in AD, and compared it with APP immunoreactivity. There was a considerable overlap of APP and APLP1 regional expression patterns. Significant APLP1 immunoreactivity was observed in neuritic plaques. Large pyramidal neurons of the subiculum showed an accumulation of APLP1 protein in their dendritic compartment. Some astrocytes elicited perinuclear APLP1 staining, but this was observed in both AD and control brains. These findings raise the possibility that APLP1 may contribute to the pathogenesis of AD-associated neurodegeneration.

Identification of cis-Elements Regulating Exon 15 Splicing of the Amyloid Precursor Protein Pre-mRNA

Alternative splicing of exon 15 of the amyloid precursor protein (APP) pre-mRNA generates two APP isoform groups APPex15 (containing exon 15) and L-APP (without exon 15), which show a cell-specific distribution in non-neuronal cells and neurons of rat. Both APP isoforms differ in regard to functional properties like post-translational modification, APP secretion, and proteolytic production of Aβ peptide from APP molecules. Since Aβ generation is an important factor in the development of Alzheimers disease, one could anticipate that these major APP isoforms might contribute differentially to the mechanisms underlying neurodegeneration in Alzheimers disease. In this study, we established an APP minigene system in a murine cell system to identifycis-acting elements controlling exon 15 recognition. A 12.5-kilobase pair genomic fragment of the murine APP gene contained all cis-regulatory elements to reproduce the splicing pattern of the endogenous APP transcripts. By using this approach, two intronic cis-elements flanking exon 15 were identified that block the inclusion of exon 15 in APP transcripts of non-neuronal cells. Point mutation analysis of these intronic regions indicated that pyrimidine-rich sequences are involved in the splice repressor function. Finally, grafting experiments demonstrated that these regulatory regions cell-specifically enhance the blockage of a chimeric exon in the non-neuronal splicing system.

Interaction of the Presenilins with the Amyloid Precursor Protein (APP).

The genes encoding presenilin-1 (PS1) and presenilin-2 (PS2) were identified as the genes that harbour mutations that cause more than 60% of early onset familial Alzheimers disease cases (FAD) (1-3). So far, more than 40 missense mutations have been described for presenilin-1 and two have been found in the gene coding for presenilin-2 (reviewed in refs. 4 and 5). Carriers of mutated presenilin genes develop in their brain neuropathological changes characteristic of Alzheimers disease including the deposition of amyloid Aβ peptide. The latter is released from its cognate amyloid precursor protein (APP) by a two-step proteolytic conversion: first, proteolysis of APP by β-secretase, which releases the N-terminus of Aβ, and second, conversion of the remaining fragment by γ-secretase, which cleaves within the predicted transmembrane region of APP. This releases the C-terminus of Aβ, which may end either at position 40 or, to a lesser extent, at position 42 (reviewed in ref. 6). The latter species, Aβ(1-42), is more prone to aggregation and deposition than Aβ(1-40) and is produced at higher levels in the brains and primary fibroblasts of FAD patients carrying PS missense mutations (7). The same result was obtained when cultured cells transfected with mutated PS1 orPS2, or transgenic mice harboring missense PS1 were analyzed for the production of Aβ(1-42): in every case increased amounts of the longer Aβ(1-42) species were observed (8-10). The mechanisms by which mutations in the PS genes affect the proteolytic processing of APP by γ-secretase have not been resolved in detail. There are two possibilities by which the normal processing of APP may be disturbed: either mutations in the presenilins affect APP metabolism in an indirect way by modulation of proteases or interaction with proteins involved in APP intracellular routing, or presenilins may modulate APP processing directly through physical interactions with APP. Such a direct interaction between presenilins and APP was first demonstrated by us for PS2 (11). Later on, formation of stable complexes with APP was reported not only for PS2 but also for PS1 (12,13,13a).