NTM0299-St. George-Hyslop - Nature

© 1999 Nature America Inc. • http://medicine.nature.com

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Presenilin mutations associated with Alzheimer disease cause defective intracellular trafficking of β-catenin,a component of the presenilin protein complex M. NISHIMURA1, G. YU1, G. LEVESQUE1, D.M. ZHANG1, L. RUEL2, F. CHEN1, P. MILMAN1, E. HOLMES1, Y. LIANG1, T. KAWARAI1, E. JO1, A. SUPALA1, E. ROGAEVA1, D-M. XU1, C. JANUS1, L. LEVESQUE1, Q. BI1, M. DUTHIE1, R. ROZMAHEL3, K. MATTILA4, L. LANNFELT4, D. WESTAWAY1, H.T.J. MOUNT1, J. WOODGETT2, P.E. FRASER1 & P. ST GEORGE-HYSLOP1


1 Centre for Research in Neurodegenerative Diseases, Departments of Medicine (Neurology), Medical Biophysics, Pathology, and Pharmacology, University of Toronto, 6 Queen’s Park Crescent West, Toronto, Ontario, Canada M5S 3H2; and Department of Medicine (Division of Neurology), The Toronto Hospital, 395 Bathurst Street, Toronto, Ontario, Canada, M5S 3H2 2 Ontario Cancer Institute, Dept of Medical Biophysics and Experimental Therapeutics, University of Toronto, 610 University Avenue, Toronto, Ontario, Canada, M5G 2M9 3 Department of Pharmacology, University of Toronto, and Department of Genetics, The Hospital for Sick Children, 555 University Avenue, Toronto 4 Karolinska Institutet, Department of Clinical Neuroscience and Family Medicine, Novum, KFC, Huddinge Hospital, 141 86 Huddinge, Sweden M.N., G.Y., G.L. & D.M.Z. contributed equally to this study Correspondence should be addressed to P.S.G.-H.; email: [email protected]

The presenilin proteins are components of high-molecular-weight protein complexes in the endoplasmic reticulum and Golgi apparatus that also contain β-catenin. We report here that presenilin mutations associated with familial Alzheimer disease (but not the non-pathogenic Glu318Gly polymorphism) alter the intracellular trafficking of β-catenin after activation of the Wnt/β-catenin signal transduction pathway. As with their effect on βAPP processing, the effect of PS1 mutations on trafficking of β-catenin arises from a dominant ‘gain of aberrant function’ activity. These results indicate that mistrafficking of selected presenilin ligands is a candidate mechanism for the genesis of Alzheimer disease associated with presenilin mutations, and that dysfunction in the presenilin–β-catenin protein complexes is central to this process.

The homologous genes for presenilin 1 (PS1) and presenilin 2 (PS2) encode polytopic transmembrane proteins localized in the nuclear envelope, the endoplasmic reticulum, Golgi apparatus and some as-yet uncharacterized intracytoplasmic vesicles1–3. The physiological functions of the presenilins are unknown, but may be related to developmental signaling, apoptotic signal transduction, or processing of selected proteins, including the β-amyloid precursor protein4–8 (βAPP). Missense mutations in the presenilins are associated with autosomal dominant forms of familial Alzheimer disease1–3 (FAD). One biochemical effect consistently associated with these mutations is an alteration in the proteolytic cleavage of βAPP such that there is overproduction of long-tailed β-amyloid peptide derivatives9–11 (for example, Aβ42). It has been speculated that this apparently dominant effect of PS1 and PS2 mutations arises either from mistrafficking of βAPP itself (although no direct evidence for this has been found)12, or from defective trafficking and/or activation of proteins involved in the proteolytic cleavage of βAPP (ref. 8). Both PS1 and PS2 are components of independent high-molecular-weight (≥250 kDa) membrane-bound complexes that are mostly found in the endoplasmic reticulum (ER) and Golgi apparatus13,14. These presenilin complexes also contain β-catenin and probably other proteins that have not been identified14,15. These 164

complexes represent an important functional presenilin moiety that may be affected by presenilin mutations. Most of the presenilin proteins (and especially the long-lived endoproteolytic fragments) are contained within these high-molecular-weight protein complexes13,14. Moreover, inclusion of mutant monomeric holoproteins and proteolytic fragments into the complexes is required for an effect on Aβ production, and unincorporated mutant proteolytic fragments are rapidly degraded16,17. Finally, perturbation of interactions between components of multimeric protein complexes is a well-accepted mechanism for both dominant ‘gain of aberrant function’ and dominant ‘loss of function’ effects of disease causing mutations. Thus, the presenilin–β-catenin complexes might be functionally compromised by mutations that cause AD and defects in βAPP processing. Given a putative role for presenilins and their Caenorhabditis elegans homologs in intracellular protein trafficking18, we have investigated the effects of presenilin mutations on the presenilin–β-catenin interaction by monitoring the intracellular trafficking of β-catenin. To achieve this, we have taken advantage of the fact that the intracellular distribution of β-catenin is regulated by activation of the Wnt/β-catenin signal transduction pathway, which can easily be manipulated pharmacologically. Our data show that presenilin mutations associated with NATURE MEDICINE • VOLUME 5 • NUMBER 2 • FEBRUARY 1999


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AD cause a perturbation in the intracellular trafficking of β-catenin. This result establishes that aberrant intracellular protein trafficking of at least selected presenilin ligands is one consequence of presenilin mutations, and indicates that functional perturbations of the multimeric presenilin complexes, a classic mechanism for dominant mutations, is likely to be intimately involved in the pathogenesis of AD. Effects of PS1 and PS2 mutations on β-catenin trafficking In response to activation of the Wnt/β-catenin signal transduction pathway and certain other stimuli, β-catenin undergoes translocation to the nucleus through a transport mechanism independent of nuclear localization signals and importin/karyopherin19. Wnt signal transduction is mediated in part by inhibition of glycogen synthase kinase-3 (GSK3), which regulates the stability of β-catenin20. In Xenopus embryos, in Dictyostelium, and in Drosophila melanogaster cells, the phenotypic effects of activation of the Wnt signal pathway can be mimicked by low concentrations of lithium ions21–23. This activity specifically arises from the inhibition of GSK3 by Li+, and has been widely used to dissect the biochemistry of downstream elements in the Wnt signalling pathway22,23. To assess the effects of presenilin mutations on nuclear translocation of β-catenin, we therefore used Li+ to activate the Wnt/β-catenin signal transduction pathway in cells expressing mutant or wild-type presenilin proteins. This method circumvents technical problems associated with the use of recombinant Wnt/wg, including the poor solubility of recombinant vertebrate Wnt proteins; the potential confounding effects of presenilin mutations on Wnt/wg receptor processing; and the absence of Wnt/wg (frizzled) receptors in many cell types. Lithium-induced nuclear β-catenin translocation was assessed by immunocytochemical measurements of endogenous nuclear β-catenin in native human fibroblasts obtained from normal control subjects and from heterozygous carriers of PS1 or PS2 mutations associated with FAD. No differences were apparent between the basal levels of endogenous nuclear β-catenin in wild-type and mutant fibroblasts (P > 0.05; Fig. 1a and e). However, after stimulation of the Wnt/β-catenin signal transduction pathway by incubation with 20 mM lithium chloride for about 3 hours, normal human fibroblasts showed the expected pronounced nuclear translocation of endogenous β-catenin (β-catenin in 364 of 474, 299 of 350, and 280 of 327 nuclei of three different control fibroblast lines co-stained with Hoechst NATURE MEDICINE • VOLUME 5 • NUMBER 2 • FEBRUARY 1999

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e β-catenin-positive nuclei (%)


Fig. 1 a, Endogenous β-catenin in mock-treated native human fibroblasts has a diffuse but predominantly cytoplasmic distribution with stronger signals beneath intercellular contact zones. Open arrows, nuclei lacking βcatenin. b, After lithium chloride treatment of wild-type fibroblasts, β-catenin is strongly localized in nuclei (filled arrows). c and d, In heterozygous PS1 mutant fibroblasts, lithium induces very little nuclear translocation (c, Ala260Val PS1; d, Met146Leu). Open arrows, nuclei lacking β-catenin; filled arrows, β-catenin localized in nuclei. e, Frequency plots of basal (black bars) and Li+-stimulated (grey bars) nuclear β-catenin immunoreactivity (scored as present or absent) in native fibroblasts from three independent normal controls (C1–3), from a heterozygous carrier of the non-pathogenic PS1 Glu318Gly polymorphism, and from heterozygous carriers of PS1 or PS2 mutations. After LiCl treatment, cells expressing endogenous mutant PS1 or PS2 show a significantly smaller increase in nuclear β-catenin compared with the ‘pooled’ wild-type controls (*, P < 0.05; **, P < 0.001). Nuclear translocation is less affected by PS2 than PS1 mutations (#, P < 0.001). Error bars are s.e.m. for at least three separate experiments; >100 cells were counted in each experiment.

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33342 dye and anti-β-catenin antibodies; Fig. 1b and e). In contrast, nuclear translocation of β-catenin was significantly reduced in human fibroblasts from heterozygous carriers of pathogenic PS1 and PS2 mutations. Thus, nuclear β-catenin was found in only 87 of 349 nuclei of PS1 Met146Leu fibroblasts; in 176 of 383 nuclei of PS1 His163Tyr fibroblasts; in 88 of 337 nuclei of PS1 Ala260Val fibroblasts; and in 69 of 370 nuclei of PS1 Leu286Val fibroblasts (χ2 = 31.9; 1 d.f. (degree of freedom); P < 0.001 for each mutation compared with control; Fig. 1c–e). Lithium-induced nuclear translocation of β-catenin was also significantly reduced in the PS2 Met239Val mutant fibroblasts (285 of 456 nuclei containing β-catenin; χ2 = 9.5; 1 d.f.; P < 0.05 compared with controls; Fig. 1e). In addition, nuclear translocation was more affected in the group of mutant PS1 fibroblasts than in mutant PS2 fibroblasts (χ2 = 29.6; 1 d.f.; P < 0.05). This observation, although singular, is in accordance with the reduced clinical penetrance and the later age of disease onset in families with PS2 mutations (mean ~ 65 years compared with ~45 years for PS1 mutations)24. In both normal and mutant cells, nuclear β-catenin staining was either robust or absent after Li+ stimulation. The explanation for this result, which has been observed in other cell types (J.W., unpublished), is not immediately apparent, but does not alter the conclusions here. To confirm that the effect of PS1 missense mutations was a specific effect of pathogenic amino acid substitutions and did not occur with non-pathogenic substitutions, we repeated these experiments in fibroblasts from a heterozygous carrier of the PS1 165


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b

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Glu318Gly polymorphism (which is not associated with increased risk for AD or with abnormal βAPP processing; ref. 25 and B. Dermaut et al., manuscript submitted). Both basal nuclear β-catenin levels (29 of 489 nuclei) and lithium-induced nuclear translocation (343 of 451 nuclei) in the PS1 Glu318Gly fibroblasts were indistinguishable from those in normal control fibroblasts (Fig. 1e). To show that these results were not an idiosyncrasy of cultured fibroblasts, we estimated endogenous nuclear β-catenin levels by western blotting of nuclear fractions from HEK293 cells stably transfected with either mutant or wild-type human presenilin cDNAs. In basal conditions, endogenous nuclear β-catenin levels were similar in native HEK293 cells and in HEK293 cells stably transfected with wild-type or mutant PS1 or PS2 cDNAs (Fig. 2a–c). After treatment with Li+, nuclear β-catenin levels substantially increased both in HEK293 cells expressing endogenous presenilins and in HEK293 cells stably expressing transfected wild-type human PS1 and PS2 (endogenous PS1/PS2: 6.8 ± 0.49fold increase; transfected wild-type PS1: 6.9 ± 0.56-fold increase; transfected wild-type PS2: 6.9 ± 0.34 fold increase, P = not significant; Fig. 2a and c). However, in HEK293 cells expressing transfected mutant human presenilin cDNAs, the lithium-induced increase in nuclear β-catenin was significantly attenuated (PS1 Leu392Val: 4.4 ± 0.62-fold increase, P < 0.03 compared with either untransfected or wild-type-transfected HEK293 cells; PS1 ∆290-319: 3.5 ± 1.00-fold, P 100 nuclei scored in each experiment.

increased affinity. However, it is clear that the estimated molecular weights of the presenilin complexes are greater than the arithmetic sum of the molecular weights of the presenilins and β-catenin, indicating that the complexes are likely to contain additional components14. Thus, such putative dysfunction of presenilin complexes is probably more likely to arise from perturbations in the interactions of these other components. Functional perturbations in multimeric complexes is a wellaccepted mechanism for both dominant ‘loss of function’ and dominant ‘gain of function’ effects. The suggestion that mutant presenilin–β-catenin complexes play a part in the pathogenesis of FAD associated with presenilin mutations is in accordance with other observations. The presenilin–β-catenin complexes are predominantly localized in the ER and Golgi13,14; presenilin mutations are associated with increased production of Aβ42 (refs. 9–11); the ER–cis Golgi network is a prominent site in neurons for the production of Aβ42 (refs. 29–31); and overexpression of Wnt in PC12 cells (which would have the opposite effect on β-catenin translocation as that of presenilin mutations) stimulates relative overproduction of Aβ40 (K. Kosik, personal communication). The functional interactions between GSK3, β-catenin and the presenilins potentially place the presenilin–β-catenin complexes within several biochemical pathways relevant to neurodegeneration. For example, GSK3 not only regulates the intracellular distribution of β-catenin, but is also thought to be involved in both the hyper-phosphorylation of Tau during the genesis of neurofibrillary tangles32, and the regulation of some aspects of βAPP processing33. Indeed, PS1 mutations may directly modulate GSK3 activity and promote hyperphosphorylation of Tau34. However, neither our own data nor that of at least one other report support this idea of a direct effect of PS1 mutations on GSK3 activity35. Disturbances in the intracellular distribution of β-catenin, however, could have several relevant effects. Thus, β-catenin/armadillo is a multi-functional protein with documented roles in signal transduction for Wnt/wg and possibly other trophic factors20, in the regulation of apoptosis in D. melanogaster neurons (which has also been postulated as a function of the presenilins)7,36, and in the integrity of synaptic junctions37. Defective regulation of these β-catenin activities could play a part in the pathogenesis of AD. However, our observations here also indicate a simpler possibility. The aberrant trafficking of β-catenin might reflect the existence of similar perturbations in trafficking or activation of other components of the presenilin complexes, which in turn modulate βAPP processing through γ-secretases8. This would provide an explanation for the failure of γ-secretase activity in PS1–/– cells (because the complex does not exist), for 168

β-cateni-positive nuclei (%)

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aberrant activity in PS1 mutant cells (because of the putative aberrant activity of the complex), and for inconsistent reports of co-immunoprecipitation of the presenilins with βAPP (because a close and direct interaction between presenilins and βAPP itself would not be required). If presenilin mutations affect Wnt/β-catenin signal transduction, why are presenilin mutations not associated with defects in embryonic development? The most parsimonious explanation is that the reduction in β-catenin translocation is not complete; mammalian Wnts are redundant; and not all units signal through β-catenin38. Nevertheless, one of two subjects known to be homozygous for PS1 mutations has moderate congenital malformations39. Our data provide a new assay for the effects of presenilin mutations which complements those based on βAPP metabolism. These data also underscore the potential importance of further analysis of the presenilin complexes as an approach to defining activities of the normal and mutant presenilins. Methods β-catenin and NFκB nuclear translocation. Native fibroblasts obtained by skin biopsy from normal subjects or subjects with PS1 mutations were plated at low density on coverslips, and treated with 20 mM lithium chloride or with control medium (unsupplemented medium or 20 mM KCl) for 3 h. Concentrations of lithium chloride were established in pilot experiments that determined the minimum concentration to induce translocation of β-catenin in most cells, and are similar to doses used by others 21–23. Cells were fixed for 10 min in 2% paraformaldehyde, incubated with 5% FBS for 30 min, stained with mouse monoclonal anti-β-catenin antibody (1:500 dilution; Transduction Labs, Lexington, Kentucky) at 4 °C overnight, and counterstained with Hoechst 33342 dye (Molecular Probes, Eugene, Oregon) to label nuclei. β-catenin-positive and -negative nuclei were then directly counted in several randomly selected fields from at least three different coverslips (about 400 cells). Cell cultures, lithium chloride treatment and nuclear counting were done by separate observers ‘blinded’ to the genotype data. HEK293 cell lines stably transfected with wild-type PS1 (wt2), Leu392Val-mutant PS1 (VL31), wild-type PS2 (sw2-9), Asn141Ile mutant PS2 (sw2-VG1) (provided by Dr. D. Selkoe), and ∆290–319 mutant PS1 were treated with test medium (lithium chloride at 5 mM final concentration) or control medium (unsupplemented or 5 mM KCl)23. For unknown reasons, HEK293 cells are more sensitive to Li+ than are fibroblasts. Nuclear fractions were prepared from lithium-treated and control cells as described40. The authenticity of these fractions was proven by light microscopic morphology with DAPI staining and by showing that they did not contain cytoplasmic proteins (β-tubulin) but did contain nuclear proteins (poly ADP-ribose polymerase (PARP) and NFκB). Concanavalin ASepharose beads (Pharmacia) were used to remove contaminating cadherin-bound β-catenin, and cytoplasmic free β-catenin was then estimated as described41. The ConA-Sepharose bead method was validated in HEK293 cells by showing that western blots of the bead-captured material NATURE MEDICINE • VOLUME 5 • NUMBER 2 • FEBRUARY 1999


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contained both β-catenin and cadherin immunoepitopes, whereas the supernatants contained only β-catenin. Proteins (5 µg) from each fraction were separated by 10% SDS–PAGE, and blots were probed with mouse monoclonal antibody against β-catenin (25 ng/ml). Signals were detected by ECL (Amersham) and were quantified from the autoradiographs by the NIH Image software package. To assess nuclear translocation of NFκB, the same HEK293 cell lines were incubated with medium alone or medium supplemented with 50 ng/ml of tumour necrosis factor-α (PeproTech, Norwood, Massachusetts) for 30 min, and then processed as above. Nuclear NFκB was quantified on western blots of nuclear preparations using rabbit polyclonal antibody against the p65 subunit42 (Santa Cruz Biotechnology, Santa Cruz, California). To investigate β-catenin ubiquitin–proteasome-mediated degradation pathways, we incubated HEK293 cells in medium containing 25 µM ALLN (N-acetyl-Leu-Leu-Norleucinal; Sigma) or ALLN plus 5 mM LiCl for 0, 1, 3 or 6 h. Cells were collected, lysates were prepared as described14, and 10 µg of protein were separated by SDS–PAGE and western blotted, followed by immunodetection using ECL and a mouse monoclonal antibody against β-catenin26 (Transduction Labs, Lexington, Kentucky). Co-immunoprecipitation. Cleared lysates of HEK293 cells stably expressing mutant or wild-type PS1 were immunoprecipitated with a rabbit polyclonal antibody to the PS1 260-409 loop (1143), and the immunoprecipitates were separated by SDS–PAGE, blotted and probed with monoclonal antibodies against β-catenin as described14. Statistics. Non-parametric Freidman analysis of variance for related samples was used to evaluate differences between sampling repetitions within each fibroblast line. Differences in nuclear β-catenin before and after Li+ treatment for different cell lines were assessed by χ2 tests for independent samples using the averaged scores for controls and for each individual mutant line. The control cell lines were not different from each other, and, to minimize the number of comparisons, data from the control cell lines were ‘pooled’ for comparison with data from the mutant cell lines. To correct for multiple comparisons, the experiment-wise error was set at α = 0.05 and the comparison-wise error at α’ = 0.006. Densitometric data from the studies of HEK293 cells were analyzed using a two-tailed Student t-test for paired comparisons. Acknowledgments This work was supported by grants from the Medical Research Council of Canada, Alzheimer Association of Ontario, EJLB Foundation, Howard Hughes Medical Research Foundation, The Canadian Genetic Disease Network, Scottish Rite Charitable Foundation, the Helen B. Hunter Fellowship (G.Y.), the Peterborough-Burgess Fellowship (E.A.R.), and the University of Toronto Department of Medicine Postgraduate Fellowship (M.N.).

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