A Pathogenic Presenilin-1 Deletion Causes Abberrant Abeta 42 Production in the Absence of Congophilic Amyloid Plaques*

Harald Steinerab, Tamas Reveszbc, Manuela Neumannbd, Helmut Romige, Melissa G. Grimf, Brigitte Pesoldag, Hans A. Kretzschmard, John Hardyh, Janice L. Holtonc, Ralf Baumeisteraf, Henry Houldenij, and Christian Haassak

From the a Adolf Butenandt-Institute, Department of Biochemistry, Laboratory for Alzheimer's Disease Research, Ludwig-Maximilians-University, 80336 Munich, Germany, the c Department of Neuropathology, Institute of Neurology, University College London, Queen Square, London, WC1N 3BG, United Kingdom, the d Institute of Neuropathology, Ludwig-Maximilians-University, 81377 Munich, Germany, e Boehringer Ingelheim KG, CNS Research, 55216 Ingelheim, Germany, the f Gene Center, Ludwig-Maximilians-University, 81377 Munich, Germany, the h Mayo Clinic, Jacksonville, Florida 32224, and i Neurogenetics, Institute of Neurology, Queen Square, London WC1N 3BG, United Kingdom

Received for publication, August 8, 2000, and in revised form, November 16, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Familial Alzheimer's disease (FAD) is frequently associated with mutations in the presenilin-1 (PS1) gene. Almost all PS1-associated FAD mutations reported so far are exchanges of single conserved amino acids and cause the increased production of the highly amyloidogenic 42-residue amyloid beta -peptide Abeta 42. Here we report the identification and pathological function of an unusual FAD-associated PS1 deletion (PS1 Delta I83/Delta M84). This FAD mutation is associated with spastic paraparesis clinically and causes accumulation of noncongophilic Abeta -positive "cotton wool" plaques in brain parenchyma. Cerebral amyloid angiopathy due to Abeta deposition was widespread as were neurofibrillary tangles and neuropil threads, although tau-positive neurites were sparse. Although significant deposition of Abeta 42 was observed, no neuritic pathology was associated with these unusual lesions. Overexpressing PS1 Delta I83/Delta M84 in cultured cells results in a significantly elevated level of the highly amyloidogenic 42-amino acid amyloid beta -peptide Abeta 42. Moreover, functional analysis in Caenorhabditis elegans reveals reduced activity of PS1 Delta I83/Delta M84 in Notch signaling. Our data therefore demonstrate that a small deletion of PS proteins can pathologically affect PS function in endoproteolysis of beta -amyloid precursor protein and in Notch signaling. Therefore, the PS1 Delta I83/Delta M84 deletion shows a very similar biochemical/functional phenotype like all other FAD-associated PS1 or PS2 point mutations. Since increased Abeta 42 production is not associated with classical senile plaque formation, these data demonstrate that amyloid plaque formation is not a prerequisite for dementia and neurodegeneration.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Alzheimer's disease (AD)1 is an age-dependent neurogenerative disorder. Although most AD cases occur sporadically, autosomal dominant inheritance has been recorded in numerous families (1). Mutations in four genes have been mapped to familial AD (FAD). These include the genes encoding the beta -amyloid precursor protein (beta APP), presenilin 1 (PS1), PS2 (1), and alpha 2-macroglobulin (2). Functional analysis revealed that beta APP and PS mutations affect endoproteolytic processing of beta APP in a very similar manner. In the amyloidogenic pathway, beta APP is first cleaved at the N terminus of the Abeta domain by the recently identified beta -secretase (3). This generates a membrane-retained C-terminal fragment, which is the substrate for the gamma -secretase. gamma -Secretase cleaves its substrate within the membrane, which results in the physiological secretion of Abeta (1). About 90% of secreted Abeta terminates at amino acid 40 (Abeta 40), while most of the remaining Abeta peptides are elongated by two amino acids (Abeta 42). The rare Abeta 42 appears to aggregate much faster than Abeta 40 (4, 5) and is therefore the major constituent of senile plaques (6, 7). FAD-associated mutations found within beta APP, PS1, and PS2 all cause the increased production of this highly amyloidogenic Abeta variant and therefore increase the kinetics of Abeta aggregation and of its deposition in congophilic senile plaques (1).

PS proteins not only affect the gamma -secretase cleavage in FAD cases but are also required for physiological Abeta generation, since a PS1 ablation results in a dramatically reduced Abeta production (8). Moreover, mutagenesis of two critical aspartate residues located within transmembrane domains 6 and 7 (TM6 and -7) also results in an inhibition of Abeta generation (9). Similar mutations in human PS2 also reduce Abeta generation (10, 11), and the critical aspartate residues are functionally conserved during evolution (12). In all cases, inhibition of PS function not only reduced Abeta generation but also concomitantly increased the corresponding membrane-retained beta APP C-terminal fragments, which are the immediate precursors for Abeta generation. Since two critical aspartate residues are required within the catalytic center of aspartyl proteases and since gamma -secretase function can be blocked by aspartyl protease inhibitors (13), it was recently claimed that PS proteins may be identical with the gamma -secretase (14).

PS proteins not only support the intramembraneous endoproteolysis of beta APP but are also required for the similar cleavage of Notch (10, 15-17). The endoproteolytic cleavage of Notch appears to be required for the generation of the Notch intracellular cytoplasmic domain (18), which translocates to the nucleus, where it is involved in transcriptional regulation (19). A function of PS in Notch signaling is also supported by the phenotypes observed in various PS1/PS2 deletions in mice (20-23), which resemble that observed upon the deletion of the Notch gene. Moreover, several mutant alleles of the Caenorhabditis elegans PS homolog sel-12 cause an egg-laying phenotype, which is due to a functional deficit in Notch signaling (24). The failure in Notch signaling in worms can be functionally rescued by transgenic expression of human PS1 or PS2 (10, 25, 26).

FAD-associated PS mutations occur frequently within the PS1 gene and are associated with the most aggressive AD phenotype (27). Out of the numerous PS mutations described to date, only three deletions (28-31) have been observed so far. However, none of the deletions are directly associated with a pathological function. We have shown previously that the pathological activity of the PS1 Delta exon9 splicing mutation (28) is independent of the large deletion and rather due to a single amino acid exchange at the aberrant splice junction at codon 290 (32). A genomic deletion of the exon 9-encoded domain (Delta exon9 Finn; see below) was reported as well (30). However, due to the aberrant splicing of exon 8 with exon 10, the same amino acid exchange is introduced at codon 290 as observed in the original PS1 Delta exon9 splicing mutation. Therefore, the amino acid sequence of PS1 Delta exon9 Finn is identical to the PS1 Delta exon9 splicing mutation. In analogy to the PS1 Delta exon9 splicing mutation (32), it would therefore be expected that this mutation (PS1 Delta exon9 Finn) produces Abeta 42 independent of the exon 9 deletion. The unusual genomic exon 9 deletion of PS1 in the Finnish pedigree is associated with Alzheimer's disease and spastic paraparesis. In contrast to all other AD cases, these patients as well as the patients with the PS1 Delta exon9 splicing mutation develop "cotton wool" plaques, which lack a congophilic dense core and plaque-related neuritic pathology (30).2 Finally, the deletion produced by the intron 4 mutation of PS1 could not be associated with an increased Abeta 42 production (29). It rather turned out that a single amino acid insertion, which is generated by aberrant splicing, is responsible for the pathological activity of this mutation (29). Therefore, no PS1 deletion has so far been associated with increased Abeta 42 generation.

We have now analyzed the function of a novel PS1 deletion (PS1 Delta I83/Delta M84; Fig. 1), which is also associated with early onset AD and spastic paraparesis. A potentially pathological function in Abeta generation and Notch signaling was specifically investigated. We found that PS1 Delta I83/Delta M84 causes increased Abeta 42 production like all other FAD-associated PS1/PS2 mutations. The PS1 Delta I83/Delta M84 mutation is associated with Abeta deposition in noncongophilic cotton wool plaques, widespread cerebral amyloid angiopathy, neurofibrillary tangles, and neuropil threads, although tau-positive abnormal neurites are rare.


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Antibodies-- Antibody 3926 to synthetic Abeta was described before (33). The polyclonal and monoclonal antibodies against amino acids 263-407 of PS1 (3027; BI.3D7) and against amino acids 297-356 of PS2 (3711; BI.HF5c) were described previously (32). For tau immunohistochemistry, the AT8 antibody (Innogenetics, Belgium) and mouse monoclonal antibody PHF1 (a gift from Peter Davies) were used. An anti-GFAP antibody (Dako, UK) was used for the detection of astrocytosis. The presence of microglia was detected with an antibody against major histocompatibility complex class II proteins (clone CR3/43), which was obtained from Dako, UK. For Abeta immunohistochemistry, N-terminal antibody 6F/3D to Abeta 8-17 (Dako, UK) or 6E10 to Abeta 1-17 (Senetek) as well as C-terminal specific antisera recognizing Abeta ending at Ala42 (antibody 44-344; Immunogenetics, Belgium) or Val40 (antibody 44-348; Immunogenetics, Belgium) were used.

Histology and Immunohistochemistry-- Brains from the patient with the PS1 Delta I83/Delta M84 mutation and from a patient with a PS1 T115C mutation were collected at postmortem and fixed in 10% formalin in phosphate-buffered saline. Blocks from the major anatomical areas, including the hippocampal formation, were processed in paraffin wax. Tissue sections were stained with hematoxylin and eosin and Bielschowsky's silver impregnation methods. Congo red and thioflavine S methods were used to detect Abeta deposits in beta -sheet conformation. For immunohistochemistry, 4-, 7-, or 20-µm sections were deparaffinized in xylene and rehydrated using graded alcohols. For PHF1, AT8, and CR3/43 immunohistochemistry, sections were pretreated in a microwave oven in sodium citrate buffer for 20 min, for GFAP immunohistochemistry in trypsin for 10 min, and for Abeta , Abeta 40, and Abeta 42 immunohistochemistry in formic acid for 10 min followed by treatment in a pressure cooker in citrate buffer for 10 min. After washes in phosphate-buffered saline and 10% milk, sections were incubated with the PHF1 antibody at 4 °C or with the GFAP, AT8, CR3/43, Abeta , Abeta 40, and Abeta 42 antibodies at room temperature. Detection of antibody binding was either performed with the ABC or the alkaline phosphatase anti-alkaline phosphatase system (DAKO) according to the manufacturer's instructions. Either diaminobenzidine/H2O2 or neufuchsin was used as chromogen.

Genetic Analysis and cDNA Encoding PS1 Delta I83/Delta M84-- Genomic DNA and mRNA were extracted from blood and frozen brain, respectively. All exons of the PS1 gene were analyzed by polymerase chain reaction amplification of genomic DNA and Big Dye sequencing. Sequence analyses of PS1 exon 4 revealed a heterozygous deletion of ATCATG at codons 83 and 84 (isoleucine-methionine) of the gene. The corresponding cDNA encoding PS1 Delta I83/Delta M84 was cloned into pcDNA3.1-zeo(+) expression vector (Invitrogen).

Cell Culture and Cell Lines-- Human embryonic kidney 293 cells (K293) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 200 µg/ml G418 (to select for beta APP expression), and 200 µg/ml zeocin (to select for presenilin expression). K293 cells stably expressing PS1 Delta I83/Delta M84 were generated by transfection of K293 cells stably expressing beta APP containing the Swedish mutation (34). K293 cells stably coexpressing Swedish beta APP695 and wt PS1 or PS1 Delta exon9 were described previously (35, 36).

Analysis of PS by Combined Immunoprecipitation/Western Blotting-- Cell lysates from stably transfected K293 cells were prepared and subjected to immunoprecipitation using the polyclonal antibody 3027 to PS1 or 3711 to PS2 (32). Following gel electrophoresis, immunoprecipitated PS proteins were identified by immunoblotting using the monoclonal antibody BI.3D7 (PS1) or BI.HF5c (PS2) (32). Bound antibodies were detected by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech).

Analysis of Abeta by ELISA-- Conditioned media (2 ml) were collected from confluent K293 cells in six-well dishes for 24 h. The media were assayed for Abeta 40 and Abeta 42 according to a previously described enzyme-linked immunosorbent assay (36).

Transgenic Lines of C. elegans and Rescue Assays-- To construct a new sel-12 expression vector, a 3.0-kilobase pair fragment of cosmid C08A12 was amplified by polymerase chain reaction using primers CCC GGC TGC AGC TCA ATT ATT CTA GTA AGC and GTC TCC ATG GAT CCG AAT TCT GAA ACG TTC AAA TAA C and cloned into pPD49.26 (37). The resulting plasmid contains only nontranscribed sequences from the 5' region of the C. elegans sel-12 gene. PS1 derivatives were cloned into this vector as a BamHI/SalI fragment. Transgenic lines were established by microinjection of plasmid DNA mixtures into the C. elegans germ line to create extrachromosomal arrays (26). Four independent lines from the progeny of F2 generation animals were established. Since the sel-12(ar171) animals never lay eggs (26), rescue of the sel-12 defect can be quantified by scoring egg-laying behavior in transgenic animals (26). 50 transgenic animals of each line were analyzed for their ability to lay eggs. The numbers of eggs laid by individual transgenic animals were counted and placed into four categories: Egl+++, robust egg laying, more than 30 eggs laid; Egl++, 15-30 eggs laid; Egl+, 5-15 eggs laid; Egl-, 0-5 eggs laid.


    RESULTS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Genetic Analysis of a Family with Autosomal Dominant Early Onset AD with Spastic Paraparesis-- Several families were identified by Houlden et al. (38) with autosomal dominant early onset AD with spastic paraparesis. Here we present the detailed pathological, biochemical, and functional analysis of one of these families. Neuropathological examination of a large Scottish family (Fig. 1A) revealed the presence of large cotton wool plaques (see below) similar to those seen in the Finnish family (30, 39). Sequencing revealed the presence of an exon 4 deletion (ATC-ATG; isoleucine-methionine) of codons 83 and 84 of the PS1 gene. The mutation was not present in 100 controls. The PS1 Delta I83/Delta M84 deletion occurs within TM1 of PS1 (Fig. 1B). TM1 may be functionally important, since other mutations were previously located in that region. Interestingly, the PS1 Delta I83/Delta M84 deletion is located immediately C-terminal to the V82L mutation (40). Moreover, a third mutation has been observed in TM1, which results in the exchange of valine at position 96 to phenylalanine (41).



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Fig. 1.   A FAD-associated PS1 deletion. A, three generation family with five affected members. All affected individuals had signs of AD with spastic paraplegia; this was progressive over 6-10 years, and all cases were wheelchair bound in the later stages of the disease. Pedigree is disguised for family protection, and the numbers to the right of individual symbols refer to the age at death or current age. Arrow, sequenced cDNA of affected patient. B, schematic representation of PS1 Delta I83/Delta M84. The deletion of amino acids Ile83 and Met84 in TM1 is shown as well as neighboring point mutations associated with FAD (asterisks). The black box represents the cleavage site domain of PS1.

Deposition of Abeta 42 in Cotton Wool Plaques-- Neuropathological investigation of the PS1 Delta I83/Delta M84 case by hematoxylin/eosin staining, Bielschowsky's silver staining, and Abeta immunohistochemistry revealed the presence of widespread cotton wool plaques (Figs. 2 and 3). These plaques were most frequently found in the neocortex, hippocampus, and striatum. Cotton wool plaques appeared in the neuropil as round, eosinophilic, and strongly Abeta -positive structures often larger than 100 µm in diameter. These frequently seemed to displace other elements such as neurons, a finding readily noticeable in the hippocampus (Fig. 2, A and B). Cotton wool plaques did not generally contain amyloid, since they were negative or occasionally very weakly stained by Congo red and weakly positive with thioflavine S (Fig. 2, C-E). Cerebral amyloid angiopathy was widespread, capillaries having thickened walls, which, together with the affected arterioles, showed apple green birefringence following Congo red staining and strong fluorescence with thioflavine S (Fig. 2, C-E). Bielschowsky silver staining (Fig. 2B) and tau immunohistochemistry (Fig. 2, F and H) revealed that neurofibrillary tangle pathology was widespread in the neocortex and hippocampal formation, although the dentate fascia was spared. Fine neuropil threads were a prominent feature within the cortices, and AT8 as well as PHF1 immunohistochemistry also revealed that the cotton wool plaques contained many thread-like processes but were only rarely associated with abnormal neurites (Fig. 2, F and H). In contrast, numerous tau (Fig. 2G) and silver-positive abnormal neurites (data not shown) were seen in association with the classical plaques found in the control case with a PS1 T115C mutation (42) (Fig. 2G). GFAP immunostaining demonstrated a relatively sparse astrocytic response to the cotton wool plaques (Fig. 2I). Furthermore, no significant microglial activation (CR3/43 immunostaining) was observed in association with cotton wool plaques (Fig. 2J), in contrast to widespread activated microglia that were clustered mostly around the amyloid plaques in the PS1 T115C case (Fig. 2K).



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Fig. 2.   Neuropathological findings in a case with PS1 Delta I83/Delta M84 mutation. A, hippocampal cotton wool plaques (arrow) displacing neurons of the CA1 hippocampal subregion (Hematoxylin and eosin, X 115). B, nonneuritic cotton wool plaques in the inner molecular layer of the dentate fascia (iML) and CA4 (arrows) (Bielschowsky's silver impregnation, × 115). C and D, Congo red-positive blood vessels but negative cotton wool plaques (Congo red preparation, × 50). E, strongly thioflavine S-positive blood vessel and weakly positive cotton wool plaques in the temporal neocortex (thioflavine S, × 150). F, an antibody recognizing phosphorylated serine 202/threonine 205 epitopes of tau labels no abnormal neurites in hippocampal cotton wool plaques (G), although numerous such structures are stained in classical plaques of a FAD case with the PS1 T115C mutation (AT8 immunohistochemistry, × 200). H, no abnormal neurites but numerous neurofibrillary tangles and neuropil threads in the temporal neocortex (PHF1 immunohistochemistry, × 190). I, relatively sparse astrocytic response to cotton wool plaques in the frontal neocortex (GFAP immunohistochemistry, × 115). J, activated microglia in the temporal cortex is found around blood vessels, but not around cotton wool plaques in the PS1 Delta I83/Delta M84 case (CR3/43 immunohistochemistry, × 200). K, activated microglia clusters around classical plaques in the PS1 T115C case (CR3/43 immunohistochemistry, × 200).



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Fig. 3.   Cotton wool plaques associated with the PS1 Delta I83/Delta M84 mutation contain Abeta 42. A and B, Abeta positive cotton wool plaques and blood vessels (arrow) in the neocortex (A, 6F/3D immunohistochemistry, × 115; B, 6E10 immunohistochemistry, × 120). The cotton wool plaques are weakly positive for Abeta 40 (44-348 immunohistochemistry, × 50) (C) but strongly positive for Abeta 42 (44-344 immunohistochemistry, × 115) (D).

Deposition of Abeta species ending at position 42 is believed to be closely associated with neuritic plaque formation in previously reported cases with different PS1 mutations (6, 7). In our PS1 Delta I83/Delta M84 case, the cotton wool plaques were strongly positive not only with antibodies raised against epitopes 8-17 (6F/3D immunostaining) or 1-17 (6E10 immunostaining) of the Abeta peptide (Fig. 3, A and B), but also with an end-specific antiserum to position 42 (Fig. 3D). In contrast, the cotton wool plaques were only weakly reactive for Abeta 40, which was found to be the predominant Abeta species deposited in blood vessels (Fig. 3C). These findings show that the cotton wool plaques are predominantly composed of Abeta ending at position 42, and, since they were also positive with the 6E10 antibody recognizing amino acids 1-17 of Abeta , at least some full-length Abeta 1-42 is deposited (Fig. 3B).

Stable Expression of PS1 Delta I83/Delta M84 in Human Cell Lines-- To further prove the pathological activity of PS1 Delta I83/Delta M84 on Abeta production, we analyzed its function in a tissue culture system, which has previously been proven to be very sensitive for the detection of abnormal Abeta 42 generation caused by the expression of FAD mutant presenilins (see Ref. 43 and references therein).

As described above, cotton wool plaques were so far found to be associated with two independent FAD mutations (Delta exon9 and Delta exon9 Finn), which both affect PS1 endoproteolysis. Since the PS1 Delta I83/Delta M84 mutation also results in a very similar pathology, we first investigated endoproteolysis of this mutant PS1 variant. cDNAs encoding PS1 Delta I83/Delta M84, PS1 Delta exon9, and wt PS1 were stably transfected into human embryonic kidney 293 cells expressing Swedish mutant beta APP (34). To prove ectopic expression and endoproteolysis of the transfected PS1 derivatives, cell lysates were immunoprecipitated with antibody 3027 to the cytoplasmic loop of PS1 (32). Immunoprecipitated PS1 proteins were identified by immunoblotting using the monoclonal antibody BI.3D7 to the C terminus of PS1 (32). Consistent with previous results (44), large amounts of uncleaved PS1 holoprotein accumulated in cells expressing PS1 Delta exon9 (Fig. 4A, upper panel). Robust amounts of PS1 C-terminal fragments (CTFs) were observed in all other cell lines including those overexpressing PS1 Delta I83/Delta M84 (Fig. 4A, upper panel). This demonstrates that the novel deletion mutation does not affect endoproteolysis of PS1 like the PS1 Delta exon 9 deletion.



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Fig. 4.   Endoproteolytic cleavage, replacement of endogenous PS, and elevated Abeta 42 production by stable expression of PS1 Delta I83/Delta M84 in K293 cells. A, upper panel, cell lysates from K293 cells expressing endogenous presenilins or overexpressing the indicated PS variants were immunoprecipitated with antibodies specific to the large loop of PS1 (3027). PS1 holoproteins and PS1 CTFs were detected by immunoblotting using the monoclonal antibody BI.3D7 (to PS1). Endoproteolytic cleavage occurs in cell lines stably expressing PS1 Delta I83/Delta M84 or wt PS1. As observed before, cell lines stably expressing PS1 Delta exon9 replace endogenous PS1 and accumulate as uncleaved full-length PS proteins (32, 44). Lower panel, cell lysates from the cell lines in (A) were immunoprecipitated with antibodies specific to the large loop of PS2 (3711), and PS2 CTFs were detected by immunoblotting using the monoclonal antibody BI.HF5c (to PS2). Overexpression of all indicated PS1 variants results in efficient replacement of endogenous PS2. B, quantitation of the Abeta 42 and Abeta 40 concentrations in conditioned media of K293 cells expressing the indicated presenilin variants using a previously described highly specific enzyme-linked immunosorbent assay (36). Expression of PS1 Delta I83/Delta M84 results in a 1.5-1.8 fold increase of Abeta 42 production. Consistent with previous results, the PS1 Delta exon9 mutation causes the production of higher Abeta 42 levels than the majority of other FAD mutations (63). However, the PS1 Delta exon9 also causes the generation of cotton wool plaques (30).

Stable expression of PS derivatives results in the displacement of endogenous presenilins (44) and is a prerequisite for functional expression of exogenous PS, since PS derivatives not displacing endogenously expressed fragments are unstable and are rapidly degraded (36, 45, 46). To prove if PS1 Delta I83/Delta M84 also displaces endogenous presenilins, PS2 was immunoprecipitated using antibody 3711 (32). Precipitated PS2 derivatives were identified by immunoblotting using the monoclonal antibody BI.HF5c (32). As demonstrated in Fig. 4A (lower panel), overexpression of PS1 Delta I83/Delta M84 led to a significant displacement of endogenous PS2 CTFs. Consistent with previous results (32, 44), expression of wt PS1 and PS1 Delta exon9 also strongly reduced the accumulation of endogenous PS2 CTFs (Fig. 4B, lower panel).

PS1 Delta I83/Delta M84 Promotes Pathological Abeta 42 Generation-- After demonstrating that overexpressed PS1 Delta I83/Delta M84 efficiently replaces endogenous PS2 CTFs, we analyzed the pathological function of the deletion mutation on Abeta 42 production by using a sensitive and specific enzyme-linked immunosorbent assay (36). Conditioned media were collected from cells stably expressing wt PS1, PS1 Delta exon9, or PS1 Delta I83/Delta M84, and Abeta 42/Abeta total ratios were determined. This revealed that the PS1 Delta I83/Delta M84 mutation induces elevated levels of Abeta 42 (Fig. 4B) that are similar to those produced by other FAD-associated point mutations (47). In contrast, and consistent with previous results (32, 35), the PS1 Delta exon9 mutation increases Abeta 42 production to even higher levels.

Reduced Facilitation of Notch Signaling-- PS1 and PS2 are both required for Notch signaling (19) and functionally replace the defective C. elegans PS homolog sel-12 (10, 25, 26). We now expressed PS1 Delta I83/Delta M84 in a mutant strain of C. elegans, which lacks a functional PS homolog (sel-12(ar171)). The sel12(ar171) animals show an egg-laying defective phenotype, which is due to a defect in Notch-signaling during vulva differentiation (24). This system can be used to monitor PS function in the facilitation of Notch signaling in an in vivo rescue assay by transgenic expression of the corresponding human cDNA constructs (24, 26). We (10, 26) and others (25) have previously shown that human wt PS1 and PS2 rescue the egg laying phenotype of the mutant worm, whereas FAD-associated PS point mutations showed a reduced rescuing activity. Consistent with previous results (25, 26), transgenic expression of wt PS1 in the mutant worm lead to a rescue of the egg laying phenotype (Table I). In contrast, PS1 Delta I83/Delta M84 showed significantly less rescuing activity (Table I). Therefore, similar to all other PS1-associated FAD mutations investigated so far (25, 32), the PS1 Delta I83/Delta M84 deletion also lost activity in Notch signaling in the in vivo rescuing assay.


                              
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Table I
Reduced rescuing activity of the sel-12 egg laying defect by PS1 Delta I83/Delta M84
For 50 transgenic animals each, the numbers of progeny were counted and were grouped in the following categories: +++, over 30 progeny laid by individual animal; ++, 15-30 progeny laid; +, 5-15 progeny laid; 0-5 progeny laid. The failure to lay eggs in the transgenic animals is the consequence of mosaic expression and/or absence of the PS1 gene, whereas egg laying in a sel-12(ar171) background can only be achieved by a functional presenilin rescuing the mutant defect.



    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The PS1 Delta I83/Delta M84 mutation is to our knowledge the first FAD-associated deletion. Almost all FAD mutations described within PS1 or PS2 are point mutations exchanging single highly conserved amino acids (48, 49). Although the recently identified intron 4 mutation can result in C-terminal truncated PS1 derivatives, these are pathologically inactive and do not cause increased Abeta 42 generation (29). This is consistent with the finding that C-terminal deleted artificial presenilins containing a FAD mutation are unable to induce Abeta 42 production as well. In that regard, mutant PS fragments have been overexpressed, which correspond to the proteolytically generated N-terminal fragment. Such PS fragments consistently failed to induce Abeta 42 generation (36, 50, 51), and even very minor deletions at the C terminus of PS1/PS2 inactivate PS function (52). The lack of pathological activity of truncated presenilins appears to be due to the failure of such PS derivatives to incorporate into the functionally required PS complex (36, 45, 50, 51, 53-56). Based on these findings, it would be unexpected that large deletions in the PS sequence would indeed be associated with early onset AD. However, one of the previously identified PS1 mutations results in the deletion of the complete exon 9-encoded domain due to a splicing error (28). Since exon 9 encodes the cleavage site of PS1 (43, 44, 57), this deletion mutation is associated with a lack of endoproteolysis and consequently the accumulation of the PS1 Delta exon9 holoprotein (44). This very drastic phenotype was believed to be the cause for the pathological activity of PS1 Delta exon9 in Abeta 42 generation. However, we have shown recently that the pathological activity of PS1 Delta exon9 splicing mutation in regard of Abeta 42 generation is solely due to a single amino acid exchange of the conserved Ser290 to cysteine (32). Therefore, this demonstrates that deletions of the PS amino acid sequence have so far not been associated with a pathological function in Abeta generation. Consistent with these findings, De Jonghe et al. (29) reported that the pathological activity of the intron 4 mutation of PS1 was surprisingly associated with an amino acid insertion generated by the utilization of a cryptic splice site but not with the deleted PS1 derivatives. Therefore, the PS1 Delta I83/Delta M84 mutation is the first pathogenic deletion of presenilins, which indeed is directly associated with a malfunction in Abeta 42 production.

Although the novel PS1 Delta I83/Delta M84 mutation is the first pathogenic PS1 deletion, it is associated with a pathological phenotype similar to that first described in association with the PS1 Delta exon9 Finn mutation. The cotton wool plaques observed lack congophilia including an amyloid core and associated abnormal neurites (30). Non-neuritic parenchymal deposits in association with extensive neurofibrillary degeneration are, however, not a unique feature of variant AD as lesions resembling cotton wool plaques, but composed of different amyloidogenic peptides also occur in the BRI gene related diseases, in familial British dementia (64-66) and familial Danish dementia (67). It is of interest that the clinical phenotype of familial British dementia and familial Danish dementia also resembles that seen in variant AD with cotton wool plaques and spasticity (38). Since Abeta 42 has a great propensity to accumulate in classical senile plaques (6, 7), one would have expected that PS1 mutations associated with noncongophilic cotton wool plaques might not affect Abeta 42 generation. However, our data demonstrate that Abeta 42 generation is significantly induced in cultured cells by the PS1 Delta I83/Delta M84 mutation very similar to all other FAD associated PS mutations investigated so far. In addition, we showed immunohistochemically that the cotton wool plaques associated with the PS1 Delta I83/Delta M84 mutation are predominantly composed of Abeta 42, which is similar to that seen in classical plaques in sporadic AD and AD caused by either APP or other PS1 mutations (6, 58, 59). PS1 Delta I83/Delta M84 not only behaves in terms of Abeta 42 generation like a typical PS-associated FAD mutation but also exhibits a similar loss of function in Notch signaling in C. elegans. Therefore, the question arises whether amyloid plaques composed of Abeta 42 are the primary cause of AD and whether such amyloid plaques initiate neurodegeneration. Apparently, the lack of classical dense core congophilic plaques did not prevent the cotton wool plaque cases from developing neurological symptoms including dementia, suggesting that the potential pathological activity of Abeta 42 may be acting upstream of amyloid deposition. Although this could indicate that increased Abeta 42 production is an epiphenomenon of FAD-associated mutations, we think it is much more likely that the previously characterized protofibrils of Abeta (4, 5) may be the primary cause for the observed neurological deficits. Since Abeta 42 can be observed intracellularly (33, 60, 61), primary pathological consequences may be induced long before Abeta finally precipitates into amyloid plaques.

Our findings may also indicate that therapeutic strategies exclusively based on the reduction of the amyloid plaque burden (62) may not always be sufficient to prevent AD symptoms.


    ACKNOWLEDGEMENTS

We thank Liane Meyn, Gabi Basset, Tammaryn Lashley, and Irvna Pigur for expert experimental assistance.


    FOOTNOTES

* This work was supported by grants from the European Community and the Deutsche Forschungsgemeinschaft (to C. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

b These authors contributed equally to this work.

g Present address: Central Institute of Mental Health, Dept. of Molecular Biology, J5, 68159 Mannheim, Germany.

j Supported by a grant from the Wellcome Trust.

k To whom correspondence should be addressed: Adolf-Butenandt-Institute, Ludwig-Maximilians-University Munich, Dept. of Biochemistry, Schillerstr. 44, 80336 München, Germany. Tel.: 49-89-5996-471/472; Fax: 49-89-5996-415; E-mail: chaass@pbm.med.uni-muenchen.de.

Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M007183200

2 H. Houlden and J. Hardy, unpublished data.


    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; FAD, familial AD; beta APP, beta -amyloid precursor protein; TM, transmembrane domain; wt, wild type.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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