Alzheimer's Disease-associated Mutations Research Articles

Losing memory by protein cleavage

Alzheimer's Disease Learning and remembering changes the shape of neurons. In mouse models of Alzheimer's disease, neurons do not undergo the morphological changes induced by learning and remembering and they also have defects in calcium signaling. Tong et al. found that forms of presenilin-1 (PS1) with familial Alzheimer's disease-associated mutations excessively cleaved and inactivated the calcium sensor STIM1. The cleavage of STIM1 by PS1 may contribute to the memory loss that is characteristic of Alzheimer's disease. Sci. Signal. 9 , ra89 (2016).

Proteolytic Processing of Neuregulin 1 Type III by Three Intramembrane-cleaving Proteases

Numerous membrane-bound proteins undergo regulated intramembrane proteolysis. Regulated intramembrane proteolysis is initiated by shedding, and the remaining stubs are further processed by intramembrane-cleaving proteases (I-CLiPs). Neuregulin 1 type III (NRG1 type III) is a major physiological substrate of β-secretase (β-site amyloid precursor protein-cleaving enzyme 1 (BACE1)). BACE1-mediated cleavage is required to allow signaling of NRG1 type III. Because of the hairpin nature of NRG1 type III, two membrane-bound stubs with a type 1 and a type 2 orientation are generated by proteolytic processing. We demonstrate that these stubs are substrates for three I-CLiPs. The type 1-oriented stub is further cleaved by γ-secretase at an ϵ-like site five amino acids N-terminal to the C-terminal membrane anchor and at a γ-like site in the middle of the transmembrane domain. The ϵ-cleavage site is only one amino acid N-terminal to a Val/Leu substitution associated with schizophrenia. The mutation reduces generation of the NRG1 type III β-peptide as well as reverses signaling. Moreover, it affects the cleavage precision of γ-secretase at the γ-site similar to certain Alzheimer disease-associated mutations within the amyloid precursor protein. The type 2-oriented membrane-retained stub of NRG1 type III is further processed by signal peptide peptidase-like proteases SPPL2a and SPPL2b. Expression of catalytically inactive aspartate mutations as well as treatment with 2,2'-(2-oxo-1,3-propanediyl)bis[(phenylmethoxy)carbonyl]-l-leucyl-l-leucinamide ketone inhibits formation of N-terminal intracellular domains and the corresponding secreted C-peptide. Thus, NRG1 type III is the first protein substrate that is not only cleaved by multiple sheddases but is also processed by three different I-CLiPs.

Potential late-onset Alzheimer's disease-associated mutations in the ADAM10 gene attenuate α-secretase activity

ADAM10, a member of a disintegrin and metalloprotease family, is an alpha-secretase capable of anti-amyloidogenic proteolysis of the amyloid precursor protein. Here, we present evidence for genetic association of ADAM10 with Alzheimer's disease (AD) as well as two rare potentially disease-associated non-synonymous mutations, Q170H and R181G, in the ADAM10 prodomain. These mutations were found in 11 of 16 affected individuals (average onset age 69.5 years) from seven late-onset AD families. Each mutation was also found in one unaffected subject implying incomplete penetrance. Functionally, both mutations significantly attenuated alpha-secretase activity of ADAM10 (>70% decrease), and elevated Abeta levels (1.5-3.5-fold) in cell-based studies. In summary, we provide the first evidence of ADAM10 as a candidate AD susceptibility gene, and report two potentially pathogenic mutations with incomplete penetrance for late-onset familial AD.

Accelerated Plaque Accumulation, Associative Learning Deficits, and Up-regulation of α7 Nicotinic Receptor Protein in Transgenic Mice Co-expressing Mutant Human Presenilin 1 and Amyloid Precursor Proteins

Familial Alzheimer's disease-associated mutations in presenilin 1 or 2 or amyloid precursor protein result in elevated beta-amyloid, beta-amyloid accumulation, and plaque formation in the brains of affected individuals. By crossing presenilin 1 transgenic mice carrying the A246E mutation with plaque-producing amyloid precursor protein K670N/M671L transgenic mice (Tg2576), we show that co-expression of both mutant transgenes results in acceleration of amyloid accumulation and associative learning deficits. At 5 months of age with no detectable plaque pathology, amyloid precursor protein transgenic animals are impaired in contextual fear learning following two pairings of conditioned and unconditioned stimuli but appear normal following a more robust five-pairing training. At 9 months of age when beta-amyloid deposition is evident, these mice are impaired following both two-pairing and five-pairing protocols. Mice carrying both transgenes are impaired in contextual fear conditioning at either age. All transgenic animal groups performed as well as controls in cued fear conditioning, indicating that the contextual fear learning deficits are hippocampus-specific. The associative learning impairments are coincident with elevated alpha 7 nicotinic acetylcholine receptor protein in the dentate gyrus. These findings provide two robust and rapid assays for beta-amyloid-associated effects that can be performed on young animals: impaired contextual fear learning and up-regulation of alpha 7 nicotinic receptors.

Calsenilin Is a Substrate for Caspase-3 That Preferentially Interacts with the Familial Alzheimer's Disease-associated C-terminal Fragment of Presenilin 2

Calsenilin is a member of the recoverin family of neuronal calcium-binding proteins that we have previously shown to interact with presenilin 1 (PS1) and presenilin 2 (PS2) holoproteins. The expression of calsenilin can regulate the levels of a proteolytic product of PS2 (Buxbaum, J. D., Choi, E. K., Luo, Y., Lilliehook, C., Crowley, A. C., Merriam, D. E., and Wasco, W. (1998) Nat. Med. 4, 1177-1181) and reverse the presenilin-mediated enhancement of calcium signaling (Leissring, M. A., Yamasaki, T. R., Wasco, W., Buxbaum, J. D., Parker, I., and LaFerla, F. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8590-8593). Here, we have used cultured mammalian cells that transiently or stably express calsenilin to extend the characterization of calsenilin and of the calsenilin-PS2 interaction. We have found that calsenilin has the ability to interact with endogenous 25-kDa C-terminal fragment (CTF) that is a product of regulated endoproteolytic cleavage of PS2 and that the presence of the N141I PS2 mutation does not significantly alter the interaction of calsenilin with PS2. Interestingly, when the 25-kDa PS2 CTF and the 20-kDa PS2 CTF are both present, calsenilin preferentially interacts with the 20-kDa CTF. Increases in the 20-kDa fragment are associated with the presence of familial Alzheimer's disease-associated mutations (Kim, T., Pettingell, W. H., Jung, Y., Kovacs, D. M., and Tanzi, R. E. (1997) Science 277, 373-376). However, the finding that the production of the 20-kDa fragment is regulated by the phosphorylation of PS2 (Walter, J., Schindzielorz, A., Grunberg, J., and Haass, C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1391-1396) suggests that it is a regulated physiological event that also occurs in the absence of the familial Alzheimer's disease-associated mutations in PS2. Finally, we have demonstrated that calsenilin is a substrate for caspase-3, and we have used site-directed mutagenesis to map the caspase-3 cleavage site to a region that is proximal to the calcium binding domain of calsenilin.

Familial Alzheimer's disease-associated mutations block translocation of full-length presenilin 1 to the nuclear envelope.

A polyclonal antibody, M5, to the hydrophilic loop domain of human presenilin 1 (PS1) was prepared. Western blot and immunoprecipitation analyses showed that M5 specifically recognized the processed C-terminal fragment, but not the full-length PS1. Epitope mapping analysis revealed that the essential sequence for recognition of the C-terminal fragment by M5 is DPEAQRR (302-308). The recognition of the C-terminal fragment by M5 in a processing-dependent manner was further confirmed by competitive enzyme-linked immunosorbent assay using the synthetic peptide L281 (281-311), which contains the putative processing site and the preceding amino acids to the site. Although L281 contains the epitope sequence for M5, the maximum inhibition was only 14%. Immunocytochemistry using M5 combined with hL312, which recognizes both full-length PS1 and the C-terminal fragment, allowed us to distinguish the localization of the processed C-terminal fragment from that of full-length PS1. Confocal microscopy demonstrated that the full-length form of wild-type PS1 is preferentially located in the nuclear envelope, while the processed C-terminal fragment is mainly present in the endoplasmic reticulum (ER). However, PS1 with familial Alzheimer's disease-associated mutations could not translocate to the nuclear envelope, and both the full-length and processed mutants were co-localized in the ER.

A Loss of Function Mutation of Presenilin-2 Interferes with Amyloid β-Peptide Production and Notch Signaling

Presenilin-1 (PS1) facilitates gamma-secretase cleavage of the beta-amyloid precursor protein and the intramembraneous cleavage of Notch1. Although Alzheimer's disease-associated mutations in the homologous presenilin (PS2) gene elevate amyloid beta-peptide (Abeta42) production like PS1 mutations, here we demonstrate that a gene ablation of PS2 (unlike that of PS1) in mice does not result in a severe phenotype resembling that of Notch-ablated animals. To investigate the amyloidogenic function of PS2 more directly, we mutagenized a conserved aspartate at position 366 to alanine, because the corresponding residue of PS1 is known to be required for its amyloidogenic function. Cells expressing the PS2 D366A mutation exhibit significant deficits in proteolytic processing of beta-amyloid precursor protein indicating a defect in gamma-secretase activity. The reduced gamma-secretase activity results in the almost complete inhibition of Abeta and p3 production in cells stably expressing PS2 D366A, whereas cells overexpressing the wild-type PS2 cDNA produce robust levels of Abeta and p3. Using highly sensitive in vivo assays, we demonstrate that the PS2 D366A mutation not only blocks gamma-secretase activity but also inactivates PS2 activity in Notch signaling by inhibiting the proteolytic release of the cytoplasmic Notch1 domain. These data suggest that PS2 is functionally involved in Abeta production and Notch signaling by facilitating similar proteolytic cleavages.