Biochemical and immunocytochemical characterization of calsenilin in mouse brain.

Mutations in the presenilin 1 and 2 genes cause the majority of early onset familial forms of Alzheimer's disease. Here we describe the biochemical and immunohistochemical characterization of calsenilin, a novel calcium binding protein that we have previously shown to interact with presenilins 1 and 2, in mouse brain. The co-immunoprecipitation of endogenous calsenilin and presenilin 1 demonstrates that these proteins are physiologic binding partners. Although calsenilin has been predicted to be a soluble protein, we have found that the majority of it is tightly associated with the cytoplasmic face of intracellular membranes and that it can only be dissociated using harsh treatments such as urea. In addition, we have demonstrated that calsenilin is a developmentally regulated protein that is mainly present in the brain, where it localizes to both the hippocampus and cerebellum. Calsenilin staining co-localized with the somatodendritic marker microtubule-associated protein-2 primarily in the granular cell layer of the cerebellum, indicating that calsenilin expression is primarily neuronal. In primary cultured neurons, calsenilin immunoreactivity was observed in cell bodies as well as in some neuronal processes. Co-localization experiments using specific axonal and dendritic markers indicate that these processes were mainly axonal in nature, although a smaller subset of dendrites also appears to contain calsenilin. In summary, we have established that calsenilin and presenilin 1 can interact at physiologic levels, and that calsenilin is a developmentally regulated protein that is expressed primarily in the cerebellum and hippocampus. Although calsenilin is a soluble protein, it is tightly associated with the membrane. Finally, the expression pattern of calsenilin, which is similar to that of the presenilin(s), suggests that the common locations of these two proteins provide an opportunity for physical interaction in vivo.

Calsenilin enhances apoptosis by altering endoplasmic reticulum calcium signaling.

Calsenilin (also called DREAM and KChIP3), a member of the neuronal calcium sensor family, was isolated in a yeast two-hybrid screen using an apoptotic domain of presenilin 2 as bait. Calsenilin is a cytoplasmic protein, but interacts with the COOH-termini of both presenilin 1 and presenilin 2 at the endoplasmic reticulum and the Golgi apparatus. In this study, we have investigated calsenilin's effect on apoptosis. In stable neuroglioma cell lines, we observed that calsenilin enhances apoptosis in response to serum withdrawal or thapsigargin. Consistent with these observations, caspase and apparently calpain activities were increased during apoptosis in calsenilin-overexpressing cells. Moreover, using calcium imaging we were able to show that cells treated with thapsigargin released more calcium from intracellular stores when calsenilin was overexpressed. Taken together, these data suggest that calsenilin causes cells to be more susceptible to apoptotic triggers, possibly by altering calcium dynamics.

Calcium-regulated DNA binding and oligomerization of the neuronal calcium-sensing protein, calsenilin/DREAM/KChIP3.

Calsenilin/DREAM/KChIP3, a member of the recoverin branch of the EF-hand superfamily, interacts with presenilins, serves as a calcium-regulated transcriptional repressor, and interacts with A-type potassium channels. Here we report physicochemical characterization of calcium binding, oligomerization, and DNA binding of human calsenilin/DREAM/KChIP3. Equilibrium Ca(2+) binding measurements indicate that the protein binds 3 Ca(2+) with a dissociation constant of 14 microM and a Hill coefficient of 0.7. Dynamic light scattering and size exclusion chromatography show that the Ca(2+)-bound protein exists as a dimer at protein concentrations lower than 150 microM and forms a tetramer at concentrations above 200 microM. The Ca(2+)-free protein is a tetramer in the concentration range 20-450 microM. Isothermal titration calorimetry and dynamic light scattering indicate that the Ca(2+)-free protein tetramer binds endothermically (DeltaH = +25 kcal/mol) to four molecules of DNA derived from the downstream regulatory element (DRE) of either the prodynorphin or c-fos genes. One DRE molecule binds tightly to the protein with a dissociation constant (K(d)) of 75 nM, and the other three bind more weakly (K(d) = 640 nM). No significant DNA binding was observed for the Ca(2+)-bound protein. The N-terminal protein fragment (residues 1-70) binds nonspecifically to DRE in a Ca(2+)-independent manner, whereas a C-terminal fragment containing the four EF-hands (residues 65-256) binds DRE (K(d) = 200 nM) in a Ca(2+)-regulated and sequence-specific fashion. The C-terminal fragment is a tetramer in the Ca(2+)-free state and dissociates into dimers at saturating Ca(2+) levels.

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.

Genomic structure, expression pattern, and chromosomal localization of the human calsenilin gene: no association between an exonic polymorphism and Alzheimer's disease.

Calsenilin is a recently-identified member of the neuronal calcium sensor family. Like other members of this family, it is found in the brain and binds calcium. Calsenilin was discovered by virtue of its interaction with both presenilin-1 and -2, proteins that are involved in the etiology of Alzheimer's disease. Because calsenilin may play a role in Alzheimer's disease and other disease with alterations in calcium homeostasis, we characterized the human gene. The gene, which we localized to chromosome 2, extends over a region of at least 74 kb and includes nine exons. Interestingly, the ninth exon of calsenilin contains a highly polymorphic CA repeat, adjacent to the stop codon. In a study of Alzheimer patients and their unaffected siblings, there was no evidence of association of AD with any calsenilin allele. This CA repeat will be useful for linkage and linkage disequilibrium studies to determine whether calsenilin variants contribute to risk in other diseases.

A molecular model of Alzheimer amyloid beta-peptide fibril formation.

Polymerization of the amyloid beta (Abeta) peptide into protease-resistant fibrils is a significant step in the pathogenesis of Alzheimer's disease. It has not been possible to obtain detailed structural information about this process with conventional techniques because the peptide has limited solubility and does not form crystals. In this work, we present experimental results leading to a molecular level model for fibril formation. Systematically selected Abeta-fragments containing the Abeta16-20 sequence, previously shown essential for Abeta-Abeta binding, were incubated in a physiological buffer. Electron microscopy revealed that the shortest fibril-forming sequence was Abeta14-23. Substitutions in this decapeptide impaired fibril formation and deletion of the decapeptide from Abeta1-42 inhibited fibril formation completely. All studied peptides that formed fibrils also formed stable dimers and/or tetramers. Molecular modeling of Abeta14-23 oligomers in an antiparallel beta-sheet conformation displayed favorable hydrophobic interactions stabilized by salt bridges between all charged residues. We propose that this decapeptide sequence forms the core of Abeta-fibrils, with the hydrophobic C terminus folding over this core. The identification of this fundamental sequence and the implied molecular model could facilitate the design of potential inhibitors of amyloidogenesis.

Calsenilin: a calcium-binding protein that interacts with the presenilins and regulates the levels of a presenilin fragment.

Most early-onset familial Alzheimer disease (AD) cases are caused by mutations in the highly related genes presenilin 1 (PS1) and presenilin 2 (PS2). Presenilin mutations produce increases in beta-amyloid (Abeta) formation and apoptosis in many experimental systems. A cDNA (ALG-3) encoding the last 103 amino acids of PS2 has been identified as a potent inhibitor of apoptosis. Using this PS2 domain in the yeast two-hybrid system, we have identified a neuronal protein that binds calcium and presenilin, which we call calsenilin. Calsenilin interacts with both PS1 and PS2 in cultured cells, and can regulate the levels of a proteolytic product of PS2. Thus, calsenilin may mediate the effects of wild-type and mutant presenilins on apoptosis and on Abeta formation. Further characterization of calsenilin may lead to an understanding of the normal role of the presenilins and of the role of the presenilins in Alzheimer disease.

Controlling amyloid beta-peptide fibril formation with protease-stable ligands.

We have previously shown that short peptides incorporating the sequence KLVFF can bind to the approximately 40amino acid residue Alzheimer amyloid beta-peptide (Abeta) and disrupt amyloid fibril formation (Tjernberg, L. O., Näslund, J., Lindqvist, F., Johansson, J., Karlström, A. R., Thyberg, J., Terenius, L., and Nordstedt, C. (1996) J. Biol. Chem. 271, 8545-8548). Here, it is shown that KLVFF binds stereospecifically to the homologous sequence in Abeta (i.e. Abeta16-20). Molecular modeling suggests that association of the two homologous sequences leads to the formation of an atypical anti-parallel beta-sheet structure stabilized primarily by interaction between the Lys, Leu, and COOH-terminal Phe. By screening combinatorial pentapeptide libraries exclusively composed of D-amino acids, several ligands with a general motif containing phenylalanine in the second position and leucine in the third position were identified. Ligands composed of D-amino acids were not only capable of binding Abeta but also prevented formation of amyloid-like fibrils. These ligands are protease-resistant and may thus be useful as experimental agents against amyloid fibril formation in vivo.