Anne L. Brunkan

A presenilin dimer at the core of the gamma-secretase enzyme: insights from parallel analysis of Notch 1 and APP proteolysis.

Notch receptors and the amyloid precursor protein are type I membrane proteins that are proteolytically cleaved within their transmembrane domains by a presenilin (PS)-dependent γ-secretase activity. In both proteins, two peptide bonds are hydrolyzed: one near the inner leaflet and the other in the middle of the transmembrane domain. Under saturating conditions the substrates compete with each other for proteolysis, but not for binding to PS. At least some Alzheimers disease-causing PS mutations reside in proteins possessing low catalytic activity. We demonstrate (i) that differentially tagged PS molecules coimmunoprecipitate, and (ii) that PS N-terminal fragment dimers exist by using a photoaffinity probe based on a transition state analog γ-secretase inhibitor. We propose that γ-secretase contains a PS dimer in its catalytic core, that binding of substrate is at a site separate from the active site, and that substrate is cleaved at the interface of two PS molecules.

Presenilin function and γ-secretase activity

Alzheimers disease (AD) is the most common form of dementia and is characterized pathologically by the accumulation of β‐amyloid (Aβ) plaques and neurofibrillary tangles in the brain. Genetic studies of AD first highlighted the importance of the presenilins (PS). Subsequent functional studies have demonstrated that PS form the catalytic subunit of the γ‐secretase complex that produces the Aβ peptide, confirming the central role of PS in AD biology. Here, we review the studies that have characterized PS function in the γ‐secretase complex in Caenorhabditis elegans, mice and in in vitro cell culture systems, including studies of PS structure, PS interactions with substrates and other γ‐secretase complex members, and the evidence supporting the hypothesis that PS are aspartyl proteases that are active in intramembranous proteolysis. A thorough knowledge of the mechanism of PS cleavage in the context of the γ‐secretase complex will further our understanding of the molecular mechanisms that cause AD, and may allow the development of therapeutics that can alter Aβ production and modify the risk for AD.

Presenilin 2 familial Alzheimer's disease mutations result in partial loss of function and dramatic changes in Aβ 42/40 ratios

Gene knockout studies in mice suggest that presenilin 1 (PS1) is the major γ‐secretase and that it contributes disproportionately to amyloid β (Aβ) peptide generation from β‐amyloid precursor protein (APP), whereas PS2 plays a more minor role. Based on this and other observations we hypothesized that familial Alzheimers disease (FAD) mutations in PS2 would have a dramatic effect on function in order to have an observable effect on Aβ levels in the presence of normal PS1 alleles. Only four of the eight reported FAD mutations in PS2 have altered function in vitro suggesting that the other variants represent rare polymorphisms rather than disease‐causing mutations. In support of our hypothesis, the four verified PS2 FAD mutations cause substantial changes in the Aβ 42/40 ratio, comparable with PS1 mutations that cause very‐early‐onset FAD. Most of the PS2 mutations also cause a significant decrease in Aβ 40, APP C‐terminal fragment (CTF)γ and Notch intracellular domain (NICD) production suggesting that they are partial loss of function mutations. PS2 M239V, its PS1 homolog M233V, and other FAD mutations within transmembrane (TM) 5 of PS1 differentially affect CTFγ and NICD production suggesting that TM5 of PS are important for γ‐secretase cleavage of APP but not Notch.

Presenilin-1 protects against neuronal apoptosis caused by its interacting protein PAG.

Mutations in the presenilin-1 (PS-1) gene account for a significant fraction of familial Alzheimers disease. The biological function of PS-1 is not well understood. We report here that the proliferation-associated gene (PAG) product, a protein of the thioredoxin peroxidase family, interacts with PS-1. Microinjection of a plasmid expressing PAG into superior cervical ganglion (SCG) sympathetic neurons in primary cultures led to apoptosis. Microinjection of plasmids expressing wild-type PS-1 or a PS-1 mutant with a deletion of exon 10 (PS1dE10) by themselves had no effect on the survival of primary SCG neurons. However, co-injection of wild-type PS-1 with PAG prevented neuronal death, whereas co-injection with the mutant PS-1 did not affect PAG-induced apoptosis. Furthermore, overexpression of PAG accelerated SCG neuronal death induced by nerve growth factor deprivation. This sensitizing effect was also blocked by wild-type PS-1, but not by PS1dE10. These results establish an assay for studying the function of PS-1 in primary neurons, reveal the neurotoxicity of a thioredoxin peroxidase, demonstrate a neuroprotective activity of the wild-type PS-1, and suggest possible involvement of defective neuroprotection by PS-1 mutants in neurodegeneration.

Two domains within the first putative transmembrane domain of presenilin 1 differentially influence presenilinase and γ‐secretase activity

Presenilins (PS) are thought to contain the active site for presenilinase endoproteolysis of PS and γ‐secretase cleavage of substrates. The structural requirements for PS incorporation into the γ‐secretase enzyme complex, complex stability and maturation, and appropriate presenilinase and γ‐secretase activity are poorly understood. We used rescue assays to identify sequences in transmembrane domain one (TM1) of PS1 required to support presenilinase and γ‐secretase activities. Swap mutations identified an N‐terminal TM1 domain that is important for γ‐secretase activity only and a C‐terminal TM1 domain that is essential for both presenilinase and γ‐secretase activities. Exchange of residues 95–98 of PS1 (sw95–98) completely abolishes both activities while the familial Alzheimers disease mutation V96F significantly inhibits both activities. Reversion of residue 96 back to valine in the sw95–98 mutant rescues PS function, identifying V96 as the critical residue in this region. The TM1 mutants do not bind to an aspartyl protease transition state analog γ‐secretase inhibitor, indicating a conformational change induced by the mutations that abrogates catalytic activity. TM1 mutant PS1 molecules retain the ability to interact with γ‐secretase substrates and γ‐secretase complex members, although Nicastrin stability is decreased by the presence of these mutants. γ‐Secretase complexes that contain V96F mutant PS1 molecules display a partial loss of function for γ‐secretase that alters the ratio of amyloid‐β peptide species produced, leading to the amyloid‐β peptide aggregation that causes familial Alzheimers disease.

A domain at the C-terminus of PS1 is required for presenilinase and γ-secretase activities

The structural requirements for presenilin (PS) to produce active presenilinase and γ‐secretase enzymes are poorly understood. Here we investigate the role the cytoplasmic C‐terminal region of PS1 plays in PS1 activity. Deletion or addition of residues at the PS C‐terminus has been reported to inhibit presenilinase endoproteolysis of PS and alter γ‐secretase activity. In this study, we use a sensitive assay in PS1/2KO MEFs to define a domain at the extreme C‐terminus of PS1 that is essential for both presenilinase and γ‐secretase activities. Progressive deletion of the C‐terminus demonstrated that removal of nine residues produces a PS1 molecule (458ST) that lacks both presenilinase processing and γ‐secretase cleavage of Notch and APP substrates. In contrast, removal of four or five residues had no effect (462ST, 463ST), while intermediate truncations partially inhibited PS1 activity. The 458ST mutant was unable to replace endogenous wtPS1 in HEK293 cells. Although 458ST was able to form a γ‐secretase complex, this complex was not matured, illustrated by mutant PS1 instability, lack of endoproteolysis, and little production of mature Nicastrin. These data indicate that the C‐terminal end of PS1 is essential for Nicastrin trafficking and modification as well as the replacement of endogenous PS1 by PS1 transgenes.