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Human Molecular Genetics, 2004, Vol. 13, No. 13 1321-1331
DOI: 10.1093/hmg/ddh151
Human Molecular Genetics, Vol. 13, No. 13 © Oxford University Press 2004; all rights reserved

Partial loss of presenilins causes seborrheic keratosis and autoimmune disease in mice

Jos Tournoy1,4, Xavier Bossuyt2, An Snellinx1,4, Marleen Regent1,4, Marian Garmyn3, Lutgarde Serneels1,4, Paul Saftig5, Katleen Craessaerts1,4, Bart De Strooper1,4 and Dieter Hartmann1,4,*

1Neuronal Cell Biology Laboratory, Center for Human Genetics CB 4, 2Department of Laboratory Medicine, University Hospital and 3Department of Dermatology, Catholic University of Leuven, Herestraat 49, B-3000 Leuven, Belgium, 4Flanders Institute for Biotechnology VIB IV, Herestraat 49, B-3000 Leuven, Belgium and 5Department of Biochemistry, CAU Kiel, Olshausenstrasse 40, D-24118 Kiel, Germany

Received February 12, 2004; Revised April 1, 2004; Accepted April 26, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Presenilin (PS1) and (PS2) are the centers of {gamma}-secretase that release Aß from APP in Alzheimer's disease (AD). They cleave signaling proteins like Notch and downregulate ß-catenin to modulate Wnt signaling. Inactivation of PS1 or PS1 and PS2 causes a prenatally lethal ‘Notch phenotype,’ which has hampered investigation of PS function in adulthood seriously. We have thus turned towards PS1+/–PS2–/– mice which carry the most severe reduction of PS alleles compatible with survival, to analyze the consequences of impaired PS function especially in adulthood. In these ‘partial deficient’ mice, PS1 protein concentration is considerably lowered, functionally reflected by reduced {gamma}-secretase activity and impaired ß-catenin downregulation. Their phenotype is normal up to ~6 months, when the majority of the mice develop an autoimmune disease characterized by dermatitis, glomerulonephritis, keratitis and vasculitis, as seen in human systemic lupus erythematosus. Besides B-cell dominated infiltrates, we observe a hypergammaglobulinemia with immune complex deposits in several tissues, high-titer nuclear autoantibodies and an increased CD4+/CD8+ ratio. The mice further develop a benign skin hyperplasia similar to human seborrheic keratosis as opposed to malignant keratocarcinomata observed in skin-specific PS1 ‘full’ knockouts. A partial reduction of PS function in PS1+/–PS2–/– mice causes a novel phenotype in adulthood unrelated to the developmental defects of full knockouts. As PS1+/–PS2+/– mice remain healthy, this points towards a sharply defined minimum of PS function. Skin and immune system appear to be especially sensitive targets of impaired PS function and may need careful monitoring if {gamma}-secretase inhibitors are envisaged for treating AD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Presenilins (PSs) form the active center of the {gamma}-secretase complex (1–6) that besides amyloid precursor protein (APP) (7) cleaves several other type 1 integral transmembrane proteins like Notch receptors (8). More than 100 mutations in the Presenilin 1 (PS1) and 2 (PS2) genes cause familial Alzheimer's disease (AD) by favoring the generation of Aß42 peptides (9). PSs also downregulate the concentration of ß-catenin, thereby suppressing Wnt signaling and modulate epithelial cell adhesion by binding and cleaving E-cadherin (10–14).

The multiple roles of PSs are reflected by severe ‘knockout’ phenotypes, which are dominated by impaired Notch signaling (7,15–17). These experiments also revealed quite unequal contributions of PS1 and PS2 to {gamma}-secretase, with ~80% of the activity linked to PS1 and 20% to PS2. Accordingly, PS2 deficiency not only causes non-lethal lung hemorrhages, but also significantly aggravates the late prenatally lethal phenotype of PS1–/– mice, as severely compromised PS1/PS2–/– embryos die at E9.5 (18,19).

It is highly likely that PSs play a crucial role in adult organisms as well. Notch signaling is involved in cell fate decisions in renewing or regenerating tissues (20–24), including vascular differentiation and remodeling (25–27). Notch activation retains cells in the proliferative pool (28), whereby hyperactivation can cause malignancies (29). Only in keratinocytes, Notch induces differentiation and acts as tumor suppressor (30). Notch also determines cell fate decisions between B- and T-lymphocyte lineages (31,32) and between different subsets such as CD4+ or CD8+ cells. Also by increasing ß-catenin concentrations, PS deficiency likely has pathological consequences in adults. Mutations in both ß-catenin and adenomatosis polyposis coli (APC) protein cause a spectrum of hyperplastic or malignant lesions in intestine, liver and brain (33,34). Tcf/Lef transcription factors that are normally activated by the Wnt pathway (for which ß-catenin is a key transducer) act also in lymphocyte development (35), especially for the survival and expansion of CD4+/CD8+ thymocytes.

So far, these data are composed from in vitro experiments, developmental phenotypes and human pathology. In vivo data are still scarce, e.g. conditionally targeted mice were only published for the central nervous system (CNS), resulting in mild alterations in behavior and memory, but only little effects attributable to Notch signaling (36–38). Mice with a hypomorphic PS1 allele with ~1% of the normal expression level (39) were born at half of the Mendelian percentage, accumulated APP C-terminal fragments (CTFs) in brain and displayed a gradient of segmentation defects of the lumbosacral spine. Qian et al. (40) used hPS1 constructs with a putatively neuron-specific hThy1 promoter that variably corrected the axial phenotype in correlation to PS1 expression levels and restored {gamma}-secretase activity. Notably, non-neuronal tissues were also ‘rescued,’ indicating that the neuronal specificity of the hThy1 promoter was not maintained.

Soriano et al. (14) and Xia et al. (13) also used a Thy1-driven hPS1 construct and obtained a complete rescue, again including non-neuronal tissues, whereby PS1 remained selectively absent in keratinocytes. The resulting accumulation of cytosolic ß-catenin caused skin carcinogenesis in adulthood, the first novel PS1 phenotype unrelated to its ontogenetic effects. The dissociation of Notch- and ß-catenin-dependent PS1 functions was further confirmed by Xia et al. (41), who observed a full-blown Notch phenotype in PS1 deficient mice rescued with a hPS1 D257A construct devoid of proteolytic activity, but an initially normal development with hyperplastic skin lesions appearing postnatally in mice rescued with hPS1 lacking the ß-catenin-binding loop domain.

However, owing to the unpredictable expression patterns of hThy1-driven rescue constructs, these approaches are not informative for a systemic loss or reduction of PS function in adulthood. To characterize PS functions in adulthood and identify possible consequences of a long-term partial inhibition of {gamma}-secretase, we have studied a novel genetic model, the PS1+/–PS2–/– mouse, which bears the most severe reduction in PS alleles compatible with survival. We observe reduced PS1 protein levels, causing reduced {gamma}-secretase activity and accumulation of ß-catenin. After normal development into adulthood (18,19), the mice develop a benign skin hyperplasia strikingly similar to human seborrheic keratosis and an autoimmune phenotype exhibiting features of systemic lupus erythematosus (SLE).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Biochemical alterations in PS1+/–PS2–/– cells
A considerable reduction of PS1 protein was observed in three independent groups of primary keratinocyte and fibroblast cultures compared with wild-type controls, indicating a clear gene dosage effect on PS protein expression (Fig. 1A). Similar results were obtained with total brain extracts (Fig. 1A).



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  		Figure 1. Analysis of PS1 expression and function. In (A), wild-type or PS1+/–PS2–/– fibroblast and keratinocyte cultures as well as brain extracts were tested for protein expression from the remaining PS1 allele. Loading was controlled by protein measurement and re-staining for ß-actin. Both cell types as well as brain tissue had significantly reduced PS1 protein levels, indicating the absence of an effective compensation for the reduced gene dosage. The consequences of reduced PS1 and absence of PS2 were checked in keratinocytes and brain extracts for {gamma}-secretase activity and additionally in keratinocytes for ß-catenin turnover. In (B) we show that APP CTFs, which are a direct substrate for {gamma}-secretase, are enriched in PS1+/–PS2–/– cells and brain extracts. Likewise, the cellular concentrations of both total ß-catenin and phospho-ß-catenin are increased (C) in both cytosolic and cell membrane fraction of keratinocytes (D).

 
Decreased {gamma}-secretase activity was revealed by accumulation of APP CTFs in keratinocytes (Fig. 1B) and in total brain extracts (Fig. 1B). Also ß-catenin levels (total ß-catenin and phospho–ß-catenin) were increased in keratinocytes (Fig. 1C), affecting both membrane and cytosolic fractions (Fig. 1D).

PS1+/– PS2–/– mice develop a phenotype affecting stratified epithelia and immune system in late adulthood
Seventy-five percent (25/33) of PS1+/– PS2–/– mice aged between 6 and 18 months developed wart-like protrusions and exulcerations of the skin (predominantly in transition regions between keratinized and unkeratinized epithelium like perigenital and perioral skin) and variable swellings of the anterior neck region (Fig. 2A and B, compare with Fig. 2C). Such defects were very rare (in one out of seven PS1+/–PS2–/– mice observed) at an age between 3 and 6 months, and were absent in younger or control mice and in less severe PS deficient genotypes (e.g. PS1+/+PS2–/– or PS1+/–PS2+/–). Thus, the incidence of these lesions rose rapidly during the second half of the first postnatal year, i.e. the majority of the 75% of the affected mice developed the phenotype between 6 and 12 months of age, which then increased in severity, but not frequency, during the following 6 months. In the following, the phenotype of these ‘symptomatic’ PS1+/–PS2–/– is described in detail.



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  		Figure 2. Phenotype of PS1+/–PS2–/– mice. Adult PS1+/–PS2–/– mice feature multifocal thickenings of dyskeratinized and sometimes ulcerated skin in the perigenital and perioral region (arrows in A and B). The anterior neck region is swollen dramatically (double arrows in B) compared with age-matched wild-type animals (C) because of enlargements of cervical lymph nodes attached to the salivary glands (LN and SG in D–G), as shown by macroscopic preparations (D, F) and hemataylin and eosin (H&E)-stained paraffin sections (E, G). Likewise, the spleen of adult PS1+/–PS2–/– mice was enlarged considerably (H) with ~3-fold increase in specific organ weight. In blood samples (I), a systemic inflammatory reaction was reflected by a considerable leukocytosis and {gamma}-globulinemia. These changes were accompanied by a moderate hypoalbuminemia and elevation of the {alpha}1-globulin fraction as often seen in inflammatory disease. Data are visualized as mean±standard error of mean, with P<0.005.

 
Swelling of the ventrolateral neck (Fig. 2B and C) is because of enlarged lymph nodes associated with the salivary glands (Fig. 2D–G). The mice also showed a splenomegaly (Fig. 2H) with a 3-fold increase in spleen weight/body weight ratio when compared with age-matched controls.

Also in line with a chronic immune reaction, blood samples revealed a severe leukocytosis (13.047±2.058 versus 4.985±513 in controls, Fig. 2I) and an almost tripled concentration of serum {gamma}-globulins (12.006±2.377 versus 3.838±290), most probably due to a polyclonal hyperactivity of B-cells, as no spikes characteristic for monoclonal expansions were seen. As non-specific signs of chronic inflammation, we likewise observed a moderate decrease of albumin and increase of the {alpha}1-globulin fraction.

Keratinizing epithelia exhibit a hyperplasia related to seborrheic keratosis
The affected skin had a hyperplastic and hyperkeratotic epithelium with numerous intraepithelial keratin ‘horn’ cysts and a highly papillomatotic interface to the underlying corium. Despite the severe hyperplasia, we found no evidence of malignancy like atypical nuclear morphology or invasive growth of cells. Together with wart-like macroscopic morphology, these skin lesions (Fig. 3A and B) strikingly resemble human seborrheic keratosis. A moderate hyperplasia was also seen in the non-keratinizing epithelium of the mouth, only sparing the tongue (Fig. 3C and D), and in the stomach epithelium, where 60% of the mice presented a mild hyperkeratosis when compared with age-matched controls.



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  		Figure 3. Skin histology of PS1+/–PS2–/– mice. The perigenital and perioral cutaneous swellings corresponded histologically to human seborrheic keratosis, featuring hyperplastic papillomatotic epithelia (white arrows in B, compare with wild-type skin shown in A) with keratin cysts (black arrow in B). In non-keratinizing hairless squamous epithelia such as in the mouth, we also observed a hyperplastic and papillomatous change (C, D). In parallel, large nodular aggregates of leukocytes without follicular substructure were observed in corium and subcutaneous fat tissue (black arrows in E and F, stained with H&E and anti-CD45, respectively), accompanied by more superficially positioned disseminated infiltrates (arrowheads in F). Further immunohistological subtyping indicated the presence (besides macrophages) of predominantly B-cells (G) and a smaller fraction of T-lymphocytes (H), stained for CD45 R and CD3, respectively. Cell sorting of T-lymphocytes in peripheral blood revealed an increased CD4/CD8 ratio in partially PS deficient mice (I), shown here as mean±standard error of mean, P<0.05.

 
Reduced PS expression causes a severe autoimmune phenotype in adult mice
The skin of aged PS1+/–PS2–/– mice featured a multifocal invasion of corium and subcutis by leukocytes (predominantly lymphocytes) often forming large nodular aggregates (Fig. 3 E–H). A further subtyping revealed a mixed population consisting of B- and T-lymphocytes and macrophages as demonstrated by CD45R (B-cells, Fig. 3G), CD3 (T-cells, Fig. 3H) and F4/80 (macrophages, data not shown), with a considerable preponderance of B-cells. A major fraction of the cells expressed IL2-{alpha}-receptor, indicating their activation (data not shown). In no case was an internal architecture like those of lymph follicles observed in the aggregates. Cell sorting of peripheral lymphocytes revealed a conspicuously higher CD4/CD8 ratio in PS1+/–PS2–/– cells when compared with age-matched controls (Fig. 3I), whereby the ratio of about 1 measured here in wild-type mice closely corresponds to published data (43) for likewise aged C57/B6 mice.

As these leukocyte aggregates are strikingly similar in morphology and composition to those seen in human SLE as well as in several murine autoimmune phenotypes we investigated further criteria for a similar phenotype in partially PS deficient mice.

First, we detected immunoglobulin (Ig) deposits along the basal lamina and in the subjacent connective tissue. No deposits were found in control mice (Fig. 4A–D). The dermis was immunoreactive for complement factor C3 (Fig. 4E and F), indicating that the Ig deposits found were indeed functionally active. In most tissues investigated, but especially severe in skin and kidney, arteries were surrounded and invaded by leukocytes, as characteristically seen in human ‘leukocytoclastic’ autoimmune vasculitis (Fig. 4G and H).



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  		Figure 4. Skin and cornea histology of PS1+/–PS2–/– mice. As a further hallmark of autoimmune skin disease, we observed Ig deposits along the basement membrane (arrowheads in A and B, see D and E for the respective nuclear stains) and in the subepithelial tissue (arrows in A and B). They were accompanied by immunoreactivity for complement factor C3 (F, see E for wild-type skin). The subcutis further featured an arteriitis with the leukocyte invasion of the vessel wall stained here for CD45 (G, nuclear stain in H) as seen in autoimmune leukocytoclastic vasculitis. Also the cornea was involved in the autoimmune phenotype regularly (I–L). The stroma was thickened, exhibiting dense leukocyte infiltrates mainly in its outer half (J, K) staining for CD45 (L), accompanied by corneal neovascularization (asterisk in J). In the vast majority of cases, the corneal epithelium did not exhibit obvious alterations (compare I and J), only rarely, focal dysplasia as shown in (K) were observed.

 
In addition, a keratitis of variable severity was observed (Fig. 4I–L, all photomicrographs taken at the same magnification). Dense leukocyte infiltrates concentrated in the outer half of the corneal stroma, which was considerably thickened (compare I to J and K) and pathologically vascularized. Inconstantly, the epithelium exhibited focal dysplasia characterized by an irregular thickening and a loss of its normal stratification (K).

In the kidney, another main target of SLE-like autoimmune diseases, similar leukocyte aggregates were mostly situated in the vicinity of larger blood vessels (Fig. 5A and B). Within the parenchyma, leukocytes also invaded the glomerula (arrowheads in Fig. 5C and D). Glomerula were also a target for Ig deposits ranging from isolated patches (Fig. 5E) sometimes projecting towards the podocyte layer (arrows in E) to a complete decoration of the capillary contours (Fig. 5F). Ig deposits were again accompanied by immunoreactivity for complement factor C3 (G), not seen in control glomerula (H). These histopathological changes were accompanied by a low-level microhematuria and microproteinuria, whereas only in two out of 10 mice major pathological values for protein as well as red and white blood cells were observed. Besides kidney and skin, less constant leukocyte invasions and Ig deposits were found in liver, skeletal muscle and salivary glands, again often associated with blood vessels and stroma, but less so with the respective parenchyma.



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  		Figure 5. Kidney phenotype of PS1+/–PS2–/– mice. The kidneys were regularly characterized by densely packed CD45-positive leukocyte aggregates, mostly along the large blood vessels (A: artery, V: vein) of the renal hilus (A, B). Infiltrates also developed around smaller cortical arteries (‘A’ in C) and invaded glomerula (arrowheads in C). Arteries and glomerula of wild-type kidneys did not exhibit these alterations (‘A’ and arrowhead in D); CD45 only stained some interstitial leukocytes. Note that in both cases, blood vessels were effectively washed free of cells by the perfusion, further confirming that only cells invading the tissue are revealed here. Different degrees of Ig deposition along glomerular capillaries were observed, ranging from isolated spike-like focal deposits (arrows in E) to an almost complete decoration of vascular contours (arrowheads in F). We observed a considerable deposition of complement factor C3 in the glomerula (G), which did not occur in wild-type kidneys (H). By a standard immunofluorescence test on cultured murine wild-type fibroblasts, sera of PS1+/– PS2–/– mice exhibited high titers (>1 : 500) of anti-nuclear antibodies, resulting either in a homogeneous or punctate staining pattern (I, J, K). Further testing by ELISA assays for the most commonly involved antigens indicated that the main immunoreactivity was directed against ssDNA (L). The optical density values are shown as mean±standard error of mean, with P<0.005.

 
Demonstration of anti-nuclear autoantibodies
We reproduced a standard test for human autoantibodies, applying serum from wild-type and PS1+/–PS2–/– mice in serial dilution on cultured murine wild-type fibroblasts. We could demonstrate an intense homogeneous (Fig. 5J) or speckled (Fig. 5K) nuclear immunoreactivity in sera from the partially PS deficient mice up to a titer of at least 1 : 500. In sera derived from wild-type mice, either no or only low-titer autoreactivity was present (Fig. 5I).

In nine of the 10 PS1+/–PS2–/– mice a homogeneous staining of the nucleus with a positive staining of the chromosomes of an HEp-2 substrate was observed, whereas staining of the nucleus was seen in only two of the 10 control mice. Enzyme linked immunosorbent assay (ELISA) analysis revealed no antibodies to extractable nuclear antigens (SSA, SSB, Scl-70 and Sm/RNP), neither in the control nor in the PS1+/– PS2–/– mice. Likewise, no antibodies to dsDNA were detected by both Farr assay and Crithidia luciliae assay. However, ELISA analysis revealed increased levels of anti-ssDNA IgG antibody levels in PS1+/–PS2–/– mice (Fig. 5L), but no anti-histone antibodies.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
A partial PS knockout causes a benign skin hyperplasia
In 75% of the mice older than 6 months, partial PS deficiency causes skin lesions strikingly similar to seborrheic keratosis, the common benign wart seen frequently in the elderly human. Similar lesions were absent in young adult mice (<3 months) and rarely developed within the next 3 months, pointing towards a clear link to animal age on top of the genotype. Despite the current classification of seborrheic keratosis as a purely benign lesion (in contrast to, for example, premalignant actinic keratosis), malignant keratinocyte tumors within or attached to seborrheic keratosis lesions have been reported, causing some suspicion towards a pathogenetic link between both entities (44). It is thus intriguing that in the mouse a progressive reduction of PS function (from PS1+/–PS2–/– to the functionally more severe PS1–/–) in skin goes in parallel with the transition from benign keratosis (this paper) to skin carcinoma (13).

Both ß-catenin turnover and {gamma}-secretase activity are impaired progressively from PS1+/–PS2–/– mice to PS1 deficient mice, respectively (8,13) and are candidate pathways for skin tumorigenesis (42). Xia et al. (13,41) initially linked the skin tumors only to pathologically elevated ß-catenin levels hyperactivating the Tcf/Lef pathway. Recently Nicolas et al. (30) showed that likewise keratinocyte-specific Notch1 deficiency causes keratinocyte malignancies, which, however, differed in distribution to those observed by us and by Xia et al. (13) and also caused a different eye phenotype. Whereas these data could be taken as good evidence that indeed deregulated Wnt/ß-catenin signaling plays the dominating role in skin tumorigenesis in PS deficient mice, some as yet unexplained links between the ß-catenin/Wnt and Notch signaling pathways appear to exist, as Nicolas et al. (30) reported a considerable increase of ß-catenin in Notch1 deficient keratinocytes.

These data may have intriguing implications for human dermatology as they identify a novel set of signaling pathways involved in the genesis of the common seborrheic wart and keratinocyte carcinoma of the skin. This proposal is supported by very recent reports on changes in the Wnt/ß-catenin pathway in human basal cell carcinoma (45–47). On the ‘mouse level’, our data surprisingly indicate that a small quantitative difference of PS gene dosage apparently defines a critical threshold between a benign and a malignant skin phenotype, underscoring a possible link between seborrheic warts and skin carcinoma. Screening human skin tumors for a related primary defect, e.g. mutations in Notch, ß-catenin, APC or in the PS genes might be a promising approach for the future.

A partial PS knockout causes an autoimmune phenotype
The severe autoimmune phenotype seen in 75% of the PS1+/– PS2–/– mice reveals a new in vivo aspect of PS function. Like skin, the immune system appears to be especially sensitive to a partial loss of PS function that does not cause any of the well-known developmental defects of PS deficiency. Thereby, the overall pattern of immune complex and complement deposits, hypergammaglobulinemia with high-titer nuclear autoantibodies, dermatitis and glomerulonephritis with characteristic lymphocyte infiltrates, arteriitis and keratitis has intriguing similarities to human SLE. The mice thus reflect aspects of the final destructive pathway of SLE, i.e. the precipitation of complement-binding Ig in connective tissues, along basement membranes and especially vascular walls (48,49) starting a multifocal inflammatory reaction. In SLE, the immune complexes are derived from dysregulated antibody responses against autoantigens, mainly different nuclear components such as nucleosomes or histone proteins, which are collectively seen in >90% of the cases. Accordingly, we demonstrate high-titer reactivity against ssDNA in the serum of the mice. It should be noted that the often-cited diagnostic autoantibodies against dsDNA are seen only in 50–60% of the human cases (48).

As yet, evidence for a link between PS/{gamma}-secretase and immune system has been reported only from cell culture models where Notch signaling is operative in lineage decisions favoring CD8 over CD4 T-lymphocytes or earlier on T-over B-cells. Thus, one could assume that impaired {gamma}-secretase function as seen here reduces Notch signaling and results in an excess of B-lymphocytes and stimulatory T helper cells (50,51). Similar shifts in relative cell type proportions (including an increased CD4/CD8 ratio) also have been observed in human SLE patients (reviewed in 49). It is thus highly intriguing that in PS1+/–PS2–/– mice we likewise observe an increased proportion of CD4+ T-cells and a predominance of B-cells in peripheral lymphocyte infiltrates. Thereby, it remains to be clarified whether such shifts in cell lineage decisions alone are sufficient to create a hyperreactive immune system that in turn is sufficient over time to break self-tolerance and start disease or whether additional factors could be involved.

In summary, our data provide an in vivo readout for functions of PS dependent signaling proteins such as Notch within the immune system, which were so far only analyzed in vitro (32,50,52–55) and clearly identify a generalized autoimmune disease as an outcome of a chronically impaired PS function.

The phenotype of partially PS deficient mice helps to define the risks of {gamma}-secretase inhibitors
{gamma}-Secretase has gained considerable attention for its role in AD pathogenesis. However, development and use of {gamma}-secretase inhibitors to reduce Aß formation in AD patients (56–58) has met considerable skepticism (59) since the first PS knockout data were published, demonstrating the severe side effects of a major loss of PS function. Even a knockout of the ‘less important’ PS2 was reported by our group to cause transient pulmonal hemorrhage and subsequent lung fibrosis (18).

In this regard, PS1+/–PS2–/– mice provide a novel approach to analyze long-term partial inhibition of {gamma}-secretase in vivo. Our data indicate that the transition from the PS1+/–PS2–/– genotype [resulting in healthy mice, (18,19)] to the PS1+/–PS2–/– genotype sharply defines a critical border, below which PS gene dosage and function may not drop without facing serious risks. Further research will have to show, whether a similar sharp borderline exists in humans as well and whether it will leave a sufficiently large therapeutic window for the clinical use of {gamma}-secretase inhibitors. Our data identify skin and immune system as especially dependant on intact PS function in the adult, indicating that both will have to be critically monitored in clinical trials and therapeutic applications of such drugs. However, when discussing risks of {gamma}-secretase inhibitors, one should keep in mind that they are probably more selective than a gene knockout as they will not affect ß-catenin downregulation directly and thus not cause skin dysplasia, even if an indirect effect on skin tumorigenesis by the Notch pathway (30) cannot be fully excluded.

In summary, besides providing new data on PS/{gamma}-secretase function in the adult organism, these results identify risks associated with the use of {gamma}-secretase inhibitors in vivo. They provide the potential ‘good news’ that this phenotype only occurs when PS function drops below a fairly sharply defined threshold, so that indeed a reasonable therapeutic window for these drugs may exist.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals, fixation and tissue processing
Thirty-three PS1+/–PS2–/– and wild-type mice between 6 and 18 months were perfused with 4% paraformaldehyde (PFA) or modified Bouin's solution and tissues embedded in paraffin. PFA-fixed tissues of additional four PS1+/– PS2–/– mice and controls were snap frozen and cryosectioned.

Blood was collected from 17 PS1+/–PS2–/– and 14 wild type mice of the same age range by heart puncture and stored in ethylenediamine tetra acetic acid (EDTA) vials. Serum was isolated by incubating at 37°C for 1 h followed by a 10 min spin at 8944g.

Immunohistochemistry
Immunohistology was performed as described (18), using antibodies against CD45, CD45R/B220 (both BD Pharmingen), CD3, mouse Ig (both DAKO), F4/80 (ATCC) and complement factor C3 (ICN). Fluoresceine isothiocyanate (FITC)-labeled tyramide was used for signal detection (Perkin–Elmer). Counterstain was done with bisbenzimide (Sigma).

Primary keratinocyte culture and cell extracts
Keratinocyte cultures were prepared from 1- to 2-day-old pups as described (43), and grown on uncoated dishes in keratinocyte-SFM. Confluent cells were harvested in phophate-buffered saline (PBS) (GIBCO) with protease inhibitors (pepstatin/trasylol/EDTA). Cell pellets were lysed in buffer A (5 mM TRIS/250 mM sucrose/1 mM ethylene glycol-bis-(beta-amino-ethyl ether) N,H,N',N'-tetraacetic acid, 1% Triton X-100, pH 7.4) and extracts cleared by centrifugation. Cell extracts of fibroblasts were made according to the same procedure. Cell membranes were generated from cells resuspended in buffer A without Triton X-100 using a 8.020 mm cell cracker (EMBL) and after removing debris, membranes were recovered by high-speed centrifugation (100 000g, 1 h). Cell membranes of brain were prepared through homogenization in buffer A without Triton X-100. After low-speed centrifugation (800g, 10 min), the postnuclear supernatant was ultracentrifuged (100 000g, 1 h) and the membrane pellet resuspended in PBS. Protein concentrations were determined using the Biorad Assay.

Western blotting
Cell extracts were separated on 4–12% gels (NOVEX, San Diego, CA, USA), and western blots for PS1 and APP were made as described. Rabbit antiserum B 63.1 is similar to the previously documented antiserum B 10/4 (7) kindly provided by Dr Wim Annaert. Antibodies against ß-catenin C-terminus (Santa-Cruz) and phosphorylated ß-catenin (Ser33/37/Thr 41 and Thr41/Ser45, Cell Signaling Technology) were used at 1 : 500 and 1 : 1000, respectively. For additional loading controls, membranes were restained by anti-ß-actin (Sigma).

Analysis of autoantibodies
Serum (1 : 40) was incubated with HEp-2000 substrate (Immunoconcepts, Sacramento, CA, USA) and after washing with Alexa 488-conjugated anti-mouse IgG (Molecular Probes A-110029) at 1 : 1000.

Anti-dsDNA antibodies were detected using the Crithidia luciliae substrate (Immunoconcepts) using the Alexa fluor 488 conjugate or by Farr assay using the anti-dsDNA kit from Trinity Biotech (Wicklow, Ireland).

Antibodies to histones and ssDNA were measured using Varelisa kits from Pharmacia Diagnostics (Freiburg, Germany). Antibodies to Sm, Sm/RNP, SSA, SSB, Scl-70 and Jo-1 were determined by ELISA using the RELISA® ENA antibody screening test from Immunoconcepts. All assays were performed according to the manufacturer's procedures except for the conjugate, which was a peroxidase-conjugated goat anti-mouse IgG or IgM (Nordic immunological laboratories, Tilburg, The Netherlands). Quantification of the albumin and {gamma}-globulins was done by capillary zone electrophoresis using the Beckman Coulter Paragon 2000 instrument (Brea, CA, USA). Total protein was determined using a Roche (Mannheim, Germany) reagent kit applied on a Modular automated analyzer (Roche).


   ACKNOWLEDGEMENTS
 
We are indebted to Dr Wim Annaert, Department of Human Genetics, K.U. Leuven, for the generous supply of antibodies against PS1 and APP. We further want to thank Eric Legius and Peter Vandenberghe, Department of Human Genetics, K.U. Leuven, for helpful discussions. This work was supported by the Diadem, the IUAP, the Alzheimer's Association (Pioneer award to B.D.S. and research grant to D.H.) and the VIB (junior grant to D.H.).


   FOOTNOTES
 
* To whom correspondence should be addressed at: Neuronal Cell Biology Laboratory, Center for Human Genetics CB 4, Catholic University of Leuven, Herestraat 49, B-3000 Leuven, Belgium. Tel: +32 16345994; Fax: +32 16347181; Email: dieter.hartmann{at}med.kuleuven.ac.be


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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