Presenilin-Dependent Transcriptional Control of the Aβ-Degrading Enzyme Neprilysin by Intracellular Domains of βAPP and APLP

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Presenilin-Dependent Transcriptional Control of the

Aβ-Degrading Enzyme Neprilysin by Intracellular

Domains of βAPP and APLP

Raphaëlle Pardossi-Piquard, Agnès Petit-Paitel, Toshitaka Kawarai, Claire

Sunyach, Cristine Alves da Costa, Bruno Vincent, Sabine Ring, Luciano

D’adamio, Jie Shen, Ulrike Müller, et al.

To cite this version:

Raphaëlle Pardossi-Piquard, Agnès Petit-Paitel, Toshitaka Kawarai, Claire Sunyach, Cristine Alves

da Costa, et al..

Presenilin-Dependent Transcriptional Control of the Aβ-Degrading Enzyme

Neprilysin by Intracellular Domains of βAPP and APLP. Neuron, Elsevier, 2005, 46 (4), pp.541-554.

�10.1016/j.neuron.2005.04.008�. �hal-02360772�

of the A

␤-Degrading Enzyme Neprilysin

by Intracellular Domains of

␤APP and APLP

Raphaëlle Pardossi-Piquard,

_{Agnès Petit,}

_Introduction

Toshitaka Kawarai,

_{Claire Sunyach,}

One of the two main histopathological hallmarks in

Alz-Cristine Alves da Costa,

_{Bruno Vincent,}

heimer’s disease (AD) is the senile plaque, an

extracel-Sabine Ring,

_{Luciano D’Adamio,}

_{Jie Shen,}

lular protein deposit composed in part by fibrillar

aggre-Ulrike Müller,

_{Peter St. George Hyslop,}

gates of amyloid

β-peptides (Aβ) (

Haass and Selkoe,

and Frédéric Checler

_*

1993

). A

β is a 40–42 amino acid peptide that is

gener-1

_{Institut de Pharmacologie Moléculaire et Cellulaire}

ated from the

β-Amyloid Precursor Protein (βAPP) by

Centre National de la Recherche Scientifique

two sequential cleavages. The first of these cleavages

UMR6097 CNRS/UNSA

occurs in the extracellular domain and is mediated by

Valbonne 06560

a membrane-bound aspartyl protease termed

β-secre-France

tase (

Vassar and Citron, 2000

). The second set of

cleav-2

_{Centre for Research in Neurodegenerative Diseases}

ages occurs at residues 40–42 (termed

γ-site) and at

Department of Medicine

residues 48–52 (termed

⑀-site) within the

transmem-University of Toronto and transmem-University Health Network

brane domain of the

βAPP stub generated by

β-secre-Toronto Western Hospital Research Institute

tase. The

γ-site cleavage generates Aβ, while the

con-6 Queen’s Park Crescent

current

⑀-site cleavage generates a cytosolic stub

Toronto, Ontario M5S 3H2

referred to as ICD (

Passer et al., 2000

) or AICD (

βAPP

Canada

IntraCellular Domain). The exact role of AICD remains

3

_{Institute for Pharmacy and Molecular Biotechnology}

unclear.

University of Heidelberg

Both the

γ- and the ⑀-site cleavages are mediated by

69120 Heidelberg

presenilin (PS)-independent and dependent proteases

Germany

(

De Strooper et al., 1998; Armogida et al., 2001

). The

4

_{Albert Einstein College of Medicine}

presenilin-dependent

γ-secretase and ⑀-site proteolytic

New York, New York

activities (which are often generically collectively termed

5

_{Dipartimento di Biochimica e Biotecnologie Mediche}

γ-secretase) are dependent upon a multimeric complex

Universita’ Degli Studi di Napoli Federico II

of at least four different membrane proteins including

Napoli

Presenilin 1(PS1) or Presenilin 2 (PS2), nicastrin, Aph-1,

Italy

and Pen-2 (

Yu et al., 2000; Francis et al., 2002

). In these

6

_{Center for Neurologic Diseases}

complexes, the presenilins have been proposed as a

Harvard Medical School

novel type of transmembrane aspartyl protease bearing

Boston, Massachusetts

the catalytic core of the

γ-secretase (

Wolfe et al., 1999

).

This novel type of intramembranous proteolysis

ap-parently governs the function of

βAPP and several Type

I transmembrane proteins including Notch, cadherins,

Summary

ErbB-4, CD44, or p75

_{. Many of these proteins are}

involved in a variety of vital cellular functions such as

Amyloid

␤-peptide (A␤), which plays a central role in

intracellular signaling in development and adulthood,

Alzheimer’s disease, is generated by

presenilin-cell adhesion, presenilin-cell growth and proliferation, and kinase

dependent

␥-secretase cleavage of ␤-amyloid

precur-activities (for review see

Sisodia and St.

George-Hys-sor protein (

␤APP). We report that the presenilins

lop, 2002; Pollack and Lewis, 2005

). Thus,

γ-secretase

(PS1 and PS2) also regulate A

␤ degradation.

Preseni-cleavage of Notch releases an intracellular fragment

lin-deficient cells fail to degrade A

␤ and have drastic

called NICD (Notch IntraCellular Domain), which acts

reductions in the transcription, expression, and

activ-as a transcription factor mediating signal transduction

ity of neprilysin, a key A

␤-degrading enzyme.

Nepri-in the Notch-Delta pathway, a critical Nepri-intercellular

sig-lysin activity and expression are also lowered by

naling mechanism, during both embryonic

develop-␥-secretase inhibitors and by PS1/PS2 deficiency in

_{ment and adulthood (}

_{Kopan et al., 1996; Shen et al.,}

mouse brain. Neprilysin activity is restored by

tran-1997; De Strooper et al., 1998; Kopan and Goate, 2000

).

sient expression of PS1 or PS2 and by expression

Under normal conditions, A

β occurs as a soluble

of the amyloid intracellular domain (AICD), which is

fragment, the concentration of which is normally tightly

cogenerated with A

␤, during ␥-secretase cleavage of

_{controlled below the threshold for its self-aggregation}

␤APP. Neprilysin gene promoters are transactivated

_into

_{β sheet fibrils (}

_{Burdick et al., 1992}

_{). A}

_{β is actively}

by AICDs from APP-like proteins (APP, APLP1, and

_{degraded by several enzymes including neprilysin}

APLP2), but not by A

␤ or by the ␥-secretase cleavage

_{(NEP), insulin-degrading enzyme (IDE), and}

endothelin-products of Notch, N- or E- cadherins. The presenilin-

_{converting enzyme (ECE) (}

_{Carson and Turner, 2002}

_).

Al-dependent regulation of neprilysin, mediated by

_{though these A}

_{β-degrading enzymes have been well}

AICDs, provides a physiological means to modulate

_{characterized, very little is known about the regulatory}

A

␤ levels with varying levels of ␥-secretase activity.

_{mechanisms that govern their expression and/or}

activ-ity. Nevertheless, under normal physiological

circum-stances, the balance between the rates of production

Neuron 542

Figure 1. Neprilysin Expression and Activity Are Selectively Lowered in Presenilin-Deficient Cells

(A) Synthetic A_{β40 was incubated for various time periods with the indicated wild-type or PS-deficient cells; then Aβ-related immunoreactivity} was analyzed after 16.5% Tris-tricine electrophoresis and Western blot with WO2.

(B–J) Neprilysin activity was measured in fibroblasts (B, D, and H) or blastocyst-derived (C, F, and J) homogenates (B–D and F) or intact cells (H and J). Neprilysin corresponds to total (white bars in [D] and [F]) or phosphoramidon-sensitive (B, C, H, and J) Suc-Ala-Ala-Phe-7AMC-hydrolyzing activity. Neprilysin-like immunoreactivity was monitored in whole homogenates (E and G) or by immunohistochemical labeling on intact fibroblasts (I). Bars represent the mean ± SEM of six (D), nineteen (F), three (H), or sixteen (J) independent determinations. *p < 0.001; **p < 0.0001.

(K–N) Neprilysin (NEP)-, endothelin-converting enzyme (ECE)-, and insulin-degrading enzyme (IDE)-like immunoreactivities were monitored in homogenates of PS+/+_{and PS}−/−_{fibroblasts (K). Aminopeptidase M (L), proteasome (M), and calpain (N) activities were measured on the}

indicated intact cells (L) or in fibroblast cell homogenates (M and N). Bars in (L)–(N) represent the mean ± SEM of ten (L) or three (M and N) independent determinations.

and clearance of A

β is likely to be delicately regulated,

neprilysin activity might be modulated, either directly

or indirectly, by the presenilins. This hypothesis was

di-breaking down only in circumstances that lead to the

rectly supported by the subsequent observation that,

onset of Alzheimer’s disease. We show here that

al-in comparison with wild-type cells, PS-deficient cells

though

γ-secretase cleavage produces Aβ, the other

displayed dramatically lower levels of total

neprilysin-product of

γ-secretase cleavage (AICD) specifically

like activity, phosphoramidon-sensitive activity, and

upregulates the transcription of NEP, which in turn,

ac-neprilysin protein expression. In

Figures 1

D and 1F,

celerates the degradation of A

β. This transcriptional

comparison of the white bars reveals the difference in

signaling pathway therefore provides a simple and

ele-total neprilysin-like activity: 12450 ± 934 versus 2148 ±

gant physiological mechanism for the regulation of A

β

326 for fibroblasts (

Figure 1

D; p < 0.0001) and 15450 ±

levels following physiological activation of

γ-secretase

754 versus 11160 ± 672 for blastocysts (

Figure 1

F; p <

cleavage of

βAPP.

0.0001). Similarly, phosphoramidon-sensitive activity is

lower in PS-deficient cells: 10480 ± 771 versus 1029 ±

Results

_{219 for fibroblasts (}

_{Figure 1}

_{D; p < 0.0001) and 13730 ±}

749 versus 9149 ± 672 for blastocysts (

Figure 1

F; p <

Neprilysin Expression and Activity Are Reduced

0.0001). The same held for neprilysin protein

expres-in Cells Devoid of Presenilexpres-ins

sion: 29% ± 5.4% of control expression was observed

A

β40 immunoreactivity decreases in a time-dependent

in PS

_{fibroblasts (}

_{Figure 1}

_{E, n = 7; p < 0.0001) and}

manner upon exposure of exogenous A

β40 peptide to

51% ± 3% of control expression was observed in PS

wild-type fibroblasts and blastocysts (PS

_{and BD6,}

_{blastocysts (}

_{Figure 1}

_{G, n = 11; p < 0.0001). Note that}

Figure 1

A). This decrease could be blocked by phos-

kinetic analyses indicated that NEP activity was

sig-phoramidon (

Suda et al., 1973

), a specific inhibitor of

nificantly lower at all time points in PS-deficient

fibro-neprilysin (not shown). However, we observed that

blasts (

Figure 1

B; p < 0.0001) and blastocysts (

Figure

A

β40 was not efficiently degraded by PS-deficient fi-

1

C; p < 0.0001).

broblasts or by PS-deficient blastocysts (PS

_and

_{Because neprilysin is a typical type II}

membrane-bound peptidase (

Roques et al., 1993

), we next

exam-BD8,

Figure 1

A). These results raise the possibility that

ined neprilysin activity on the surface of intact cells,

using a cell-impermeable fluorimetric substrate. In this

assay, the substrate is cleaved only by enzymes that

are present at the cell surface with their catalytic sites

facing the extracellular space. In agreement with the

studies on whole-cell lysates described above,

PS-deficient fibroblasts exhibited a significant 80%

reduc-tion of cell membrane neprilysin activity compared to

that in wild-type fibroblasts (31130 ± 582 versus 4609

± 359,

Figure 1

H; p<0.0001). Furthermore, neprilysin

im-munoreactivity was poorly detectable at the surface of

intact PS-deficient fibroblasts, although it was readily

detectable on the surface of wild-type fibroblasts (

Fig-ure 1

I). A similar reduction in cell membrane neprilysin

activity was also observed in PS-deficient blastocysts

(BD8), but not in wild-type blastocysts (BD6) (380.7 ±

34 versus 199 ± 25,

Figure 1

J; p < 0.001).

Presenilin Deficiency Selectively Affects Neprilysin

To assess whether PS deficiency specifically altered

neprilysin activity, or whether it also affected other

pu-tative A

β-degrading activities or proteases, we

mea-sured the expression of endothelin-converting enzyme

and insulin-degrading enzyme (

Figure 1

K) and the

Figure 2. Presenilin Deficiency Does Not Affect Neprilysin at a

Post-activities of aminopeptidase M, another ectoenzyme

transcriptional Level

(

Figure 1

L) (

Checler, 1993

), proteasome (

Figure 1

M),

(A) NEP was immunoprecipitated from PS+/+_{and PS}−/−_fibroblast

and calpain (

Figure 1

N). In sharp contrast to the effects

homogenates. Immunological complexes were analyzed for their

of PS-deficiency on neprilysin, none of these other en-

PS1-zymes were affected by the absence of PS1 and PS2.

product of PS1 (CTF-PS1;Checler, 1999).

(B) Endogenous cell distribution of NEP was analyzed by sucrose

Presenilin 1 and Presenilin 2 Affect

density gradient. Note that NEP immunoreactivity is drastically

Neprilysin Transcription

lower in PS−/−_{fibroblasts (while controlβ-actin is identical) and}

The presenilins directly interact with several unrelated

proteins such as nicastrin, Aph-1, and Pen-2, and many

of these proteins are destabilized by the absence of the

prevent NEP neosynthesis as described in theExperimental

Pro-presenilins (for review, see

De Strooper, 2003

).

How-cedures. At the indicated times, NEP activity was fluorimetrically

ever, six lines of evidence indicate that the reductions

recorded. Note the identical slopes (−1.65 ± 0.22 [PS+/+_{] and}

in neprilysin in PS-deficient cells are not due to the loss

−1.35 ± 0.31 [PS−/−_{]), indicating a PS-independent similar decay of}

of a direct, stabilizing interaction between the preseni-

determin-lin and neprilysin proteins, but rather arise from a re-

duction of neprilysin transcription. First, anti-NEP im-

(D) Forty-eight hours after transfection in PS+/+_{and PS}−/−

fibro-munoprecipitates from wild-type fibroblasts do not

blasts, NEP expression (upper panel) and activity (lower panel)

contain PS1 (

Figure 2

A) or PS2 (not shown). Second,

were monitored as described in theExperimental Procedures. Bars

PS1 and PS2 immunoprecipitation does not deplete

su-represent the mean of two independent experiments carried out

pernatants of NEP activity (not shown). Third, residual

NEP expression in PS

_{fibroblasts partitioned within}

cell compartments with the same distribution as in

wild-type fibroblasts (

Figure 2

B), suggesting that PS

activity were not affected in cells devoid of either PS1

only or PS2 only. Thus, normal levels of neprilysin

deficiency did not alter trafficking of NEP in PS

fibro-blasts. Fourth, neprilysin stability is not affected by PS

mRNA (

Figure 3

A), enzymatic activity (

Figure 3

B), and

protein expression (

Figure 3

C) were observed in PS1

deficiency (

Figure 2

C). Fifth, both neprilysin expression

and cell surface neprilysin activity can be fully restored

fibroblasts (expressing only endogenous PS2) and in

PS2

_{fibroblasts (expressing only endogenous PS1).}

by transfection of neprilysin cDNA into PS-deficient

fi-broblasts (

Figure 2

D) and into PS-deficient blastocysts

Identical results were achieved when exogenous PS1

or exogenous PS2 were transfected into PS-deficient

(120% ± 4.6% above control mock-transfected cells;

p < 0.01; data not shown). Finally, quantitative RT-PCR

fibroblasts. Thus, neprilysin mRNA expression, protein

expression, and enzymatic activity were equivalently

analyses revealed an approximately 80% reduction in

neprilysin mRNA (

Figure 3

A) (note that overexposure of

and fully restored in PS-deficient fibroblasts by

tran-sient transfection of PS1 and PS2, PS1 only, or PS2

the gel [lower panel] shows residual mRNA expression).

Taken together, these data suggest that the loss of

only (

Figures 3

D and 3E). Control experiments indicate

that aminopeptidase M activity was not affected by

neprilysin expression in PS-deficient cells arises from

impairment in neprilysin transcription. Intriguingly, nep-

PS1 or PS2 complementation in PS-deficient

fibro-blasts (

Figure 3

F). These data were fully confirmed

rilysin transcription, protein expression, and enzymatic

Neuron 544

Figure 3. NEP mRNA Expression Is Reduced in PS-Deficient Cells and Restored by Either PS1 or PS2. NEP Is Reduced in Brain Tissue from Conditional Knockout Mice Lacking Both Presenilins

(A) Analysis of NEP mRNA expression by RT-PCR in PS+/+_{, PS1}−/−_{, PS2}−/−_{, and PS1}−/−_/PS2−/−_(PS−/−_{) fibroblasts. Note that only the combined}

depletion of PS1 and PS2 reduces NEP mRNA expression by 80% (lower panel, an overexposed gel analysis), while PS1 or PS2 invalidation does not affect NEP mRNA expression.

(B and C) PS1−/−_{and PS2}−/−_{fibroblasts display unaffected NEP activity (B) and expression (C). Data in (B) represent the mean ± SEM of five}

independent determinations.

(D and E) PS−/−_{fibroblasts were transiently transfected with PS1, PS2, or both (PS1/2) cDNAs, and then NEP mRNA and protein (D) or activity}

(E) was monitored. Note that PS1 or PS2 cDNA alone fully restores NEP mRNA expression as well as NEP expression and activity. Bars in (E) represent the mean ± SEM of three independent determinations.

(F) In the same PS−/−_{transfected cells, aminopeptidase M activity remains unaffected. Bars represent the mean of two independent}

determin-ations.

(G–J) PS1−/−_{(G) and double KO PS1}−/−_PS2−/−_{(H–J) mice brains were homogenized and examined for NEP activity (G and J) or expression (I).}

Bars in (H) correspond to the densitometric analysis of NEP expression in three independent determinations; *p < 0.05. Bars in (G) and (J) correspond to the mean of three independent determinations; **p < 0.01.

in vivo because PS1-deficiency did not alter brain nep-

the

γ-secretase inhibitor DAPT (

Dovey et al., 2001

) led

to a 50% inhibition of neprilysin activity (p < 0.0001,

rilysin activity (

Figure 3

G), while neprilysin expression

Figure 4

A). The inhibitory effect was maximal at 48 hr

(

Figures 3

H and 3I) and activity (

Figure 3

J) were

simi-of treatment, a time point at which DAPT was inert on

larly and significantly reduced in brain tissue from

con-aminopeptidase activity (

Figure 4

B). Other

γ-secretase

ditional double knockout mice lacking both presenilins

inhibitors, namely L685,458 (

Shearman et al., 2000

) or

(41% ± 9% and 29% ± 2.3% inhibition of NEP

expres-DFK167 (referred to as MW167 in

Wolfe et al. [1998]

)

sion and activity, respectively, n = 3; p < 0.05 in PS

also elicited significant reduction of neprilysin activity

versus control brain). These data therefore lead to the

(p < 0.001 and p < 0.05, respectively,

Figure 4

C), while

conclusion that PS1 and PS2 may have redundant roles

a control calpain inhibitor (CPI) was totally ineffective

in regulating neprilysin transcription, but depletion of

(

Figure 4

C). Importantly, DFK167 also lowers neprilysin

both PS1 and PS2 significantly reduces transcription of

activity in TSM1 neurons (30.4% ± 3.8% of inhibition

this enzyme.

versus control, n = 5; p < 0.0001,

Figure 4

E) and in

pri-mary cultured neurons (56.3% of inhibition, n = 2,

Fig-␥-Secretase Inhibitors Reduce Neprilysin Activity

ure 4

F). All inhibitors remain inactive on the residual

in Neuronal and in Wild-Type Cells but Not

neprilysin activity observed in PS-deficient fibroblasts

in PS-Deficient Fibroblasts

(

Figure 4

D) and do not affect in vitro NEP activity (not

The reduction in neprilysin transcription in PS-deficient

_shown).

cells could arise from loss of presenilin-dependent

γ-secretase activity or from loss of some other putative

AICDs Upregulate Neprilysin Activity and

activity of the presenilin complexes. To resolve this ques-

Expression in PS-Deficient Fibroblasts and

tion, we examined whether neprilysin activity could be

Blastocysts and Neprilysin Promoter Transactivation

directly modulated in wild-type cells by

γ-secretase in-

The canonical

γ-secretase-mediated hydrolysis

liber-ates the C terminus of A

β40/42 and concomitantly

re-hibitors. Chronic treatment of wild-type fibroblasts with

Figure 4. Effect ofγ-Secretase Inhibitors and γ-Secretase-Derived βAPP Fragments on NEP Activity and Promoter Transactivation

(A–F) Wild-type (PS+/+_{) fibroblasts were chronically treated by successive additions of DAPT for a total time period of 8, 24, and 48 hr (see}

Experimental Procedures) (A) or for 48 hr (B) with 2␮M of DAPT, and then NEP (A) or aminopeptidase M (B) activities were fluorimetrically recorded on intact cells. Error bars represent the mean ± SEM of three to six independent determinations. PS+/+_{(C), PS}−/−_{fibroblasts (D),}

TSM1 neurons (E), and primary cultured neurons (F) were treated with the indicated inhibitor (DAPT, 48 hr, 2␮M; L685,458, 8 hr, 1 ␮M; DFK167, 8 hr, 100_{␮M; calpain inhibitor [CPI], 8 hr, 100 ␮M), and then the NEP activity of intact cells was measured. Error bars represent the} mean ± SEM of three to five independent determinations; ***p < 0.0001.

(G–K) PS-deficient (PS−/−_{) fibroblasts or PS-deficient (BD8) blastocyst-derived cells (G) were treated for various time periods with 10 ng/ml of}

Aβ42, and then NEP activity was fluorimetrically assayed on intact cells. Error bars represent the mean ± SEM of three experiments. PS−/−

fibroblasts (H and J) or BD8 cells (I and K) were transiently transfected with empty vector or with the indicated AICD cDNA , and then NEP activity (H and I) or expression (J and K) was monitored. Bars in (H) and (I) represent the mean ± SEM of five independent experiments. (L and M) The indicated AICD was cotransfected in fibroblasts (L) or in TSM1 neurons (M) withβ-gal cDNA and either renal NEP promoters rNEPP1 and rNEPP2 (L) or neuronal NEP promoter nNEPP (M), and then_{β-galactosidase and luciferase activities were monitored.}

(N) HEK293 cells were transfected with AICDC59, Fe65, and Tip60 cDNA , and then nuclear extracts and binding experiments with the 219 bp probe derived from rNEPP were carried out as described in theExperimental Procedures. Lane 1, labeled probe + nuclear extract; lane 2, labeled probe + nuclear extract + Anti-myc; lane 3, labeled probe + nuclear extract + unrelated antibody.

leases the 59 amino acid stub composed of the cyto-

neprilysin promoter elements upstream of a luciferase

reporter minigene. Both AICDs dramatically increased

plasmic C-terminal tail of

βAPP referred to as AICDC59

(see

Introduction

and

Figure 8

). An additional prese-

the transactivation of two renal neprilysin promoters,

rNEPP1 (−385 bp to +147 bp) and rNEPP2 (−263 bp

nilin-dependent proteolytic cleavage of

βAPP and

Notch occurs several amino acids downstream (re-

to +145 bp,

Figure 4

L) in fibroblasts. To confirm

AICD-induced transactivation of the neprilysin promoter in a

ferred to as

⑀ cleavage). This ⑀ cleavage event liberates

AICDC50 from APP and a Notch IntraCellular Domain

neural cell line, we also cloned the neuronal neprilysin

promoter and repeated the luciferase reporter assay. In

(NICD) from Notch (

Gu et al., 2001; Sastre et al., 2001;

Weidemann et al., 2002

). Because NICD is known to

agreement with the above observations, AICDC50 also

transactivated neprilysin promoter in TSM1 neurons

modulate the transcription of several genes, we

rea-soned that neprilysin transcription might be modulated

(

Figure 4

M). Supergel shift assay analysis

demon-strated that the AICD-potentiated transactivation of the

by one of the

γ/⑀-secretase-derived products.

Exoge-nous A

β42 did not modify neprilysin activity in PS-defi-

neprilysin promoter indeed appears to be mediated by

a direct physical interaction of AICD with the neprilysin

cient fibroblasts or in PS-deficient blastocysts (

Figure

4

G). However, transient transfections of AICDC50 or

promoter (

Figure 4

N).

AICDC59 cDNAs increased both neprilysin activity (

Fig-ures 4

H and 4I) and neprilysin expression (

Figures 4

J

AICD-Induced Complementation of Neprilysin

Activity Is Potentiated by Fe65 and Tip60

and 4K) in PS-deficient fibroblasts and blastocysts.

In order to link our observation of AICD-induced in-

in Fibroblasts and in HEK293 Cells

Several lines of evidence have indicated that the

adap-crease of neprilysin activity and expression to our

ob-servation of PS-dependent neprilysin mRNA upregula-

tor protein Fe65 modulates the stability of AICD (

Kim-berly et al., 2001; Kinoshita et al., 2002

), thereby

po-tion, we examined the effect of AICDC50 and AICDC59

Neuron 546

Figure 5. Effect of Fe65 and Tip60 on NEP in Fibroblasts and HEK293 Cells

(A–C) PS+/+_{([A], upper panel and [C]) and PS}−/−_{(B and C) fibroblasts were transiently transfected with the indicated mix of cDNAs; then NEP,}

ECE, IDE, Fe65, andβ−tubulin expressions were measured by Western blot. (Note that gel in [B] corresponds to a long exposure in order to visualize any putative effect of Fe65 and Tip60 on residual NEP). Densitometric analyses (C) indicate that transfection of Fe65 and Tip60 cDNAs alone increases NEP expression in PS+/+_{(p < 0.0005 when compared to vector alone) but not in PS}−/−_{. Error bars in (C) represent the}

mean ± SEM of four independent determinations. (A, D, and F) HEK293 ([A], lower panel, [D], and [F]) were transiently transfected with the indicated mix of cDNAs (A) or indicated X11 cDNA (F); then NEP, ECE, IDE, Fe65, actin, AICDC50, AICDC59, and X11 expressions were measured by Western blot. In (D), AICDC59-like immunoreactivity was assessed by immunohistochemistry after transfection of AICDC59 alone (upper panels) or together with Fe65 and Tip60 (lower panels) in HEK293 cells. Note the increase of AICDC59 expression and the nuclear redistribution of AICDC59-like immunoreactivity (shown by merge with nuclear DAPI label [left panels]) triggered by Fe65 and Tip60 cDNA transfections. (E) NEP mRNA (upper panel) and activity (lower panel) are decreased by Fe65 deficiency in fibroblasts. Bars in (E) and (F) represent the mean ± SEM of four (E) or five (F) independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

interaction with the histone acetyltransferase Tip60

Tip60 (142.9% ± 19% and 174.9% ± 19% for AICDC50+

Fe65+Tip60 and AICDC59+Fe65+Tip60 versus control,

(

Cao and Südhof, 2001

). We therefore examined whether

Fe65 and Tip60 could (1) influence neprilysin by modu-

respectively, n = 3; p < 0.05). Fe65 and Tip60 (either

alone or in combination with AICDs) had no effect on

lating endogenous AICD in wild-type fibroblasts and in

HEK293 cells and (2) potentiate AICD-induced increase

the expressions of endothelin-converting enzyme and

insulin-degrading enzyme in PS

_{fibroblasts and}

in neprilysin activity.

Fe65 and Tip60 transfection enhanced neprilysin ex-

HEK293 cells (

Figure 5

A), a result that is in agreement

with the experiments described above showing that the

pression and activity in PS

_{fibroblasts (}

_{Figure 5}

_A

[up-per panel] and

Figure 5

C; p < 0.0005) and in HEK293

presenilin-dependent enhancement of A

_{β degradation}

is specific to neprilysin (see

Figure 1

). In order to

exam-cells (

Figure 5

A [lower panel]), but not in PS-deficient

cells (

Figures 5

B and 5C), indicating that Fe65 and

ine whether Fe65 could be a limiting factor for

expres-sion of neprilysin, we examined the activity of neprilysin

Tip60 augment neprilysin expression through functional

interaction with an endogenous PS-dependent prod-

in p97Fe65-deficient mice fibroblasts (

Wang et al.,

2004

). Abolition of p97Fe65 diminished neprilysin

activ-uct. In wild-type PS

_{fibroblasts, the cotransfection of}

Fe65 and Tip60 with either AICDC50 or AICDC59 cDNA

ity (52% ± 5.9% of decrease in Fe65

_{versus control}

fibroblasts, n = 4; p < 0.01) (

Figure 5

E) and mRNA

ex-increased neprilysin expression when compared to

AICDs cDNA transfection alone (

Figure 5

A). This was

pression (27% ± 1.5% of decrease in Fe65

_versus

control fibroblasts, n = 3; p < 0.005) (

Figure 5

E).

accompanied by an augmentation of AICDC50 and

AICDC59 immunoreactivities (

Figure 5

A [lower panel])

Interestingly, the overexpression of X11

α, X11β, and

X11

γ that triggers opposite effects on Aβ recovery

and by a clear translocation of AICDC59 (

Figure 5

D)

and AICDC50 (not shown) into the nuclei of HEK293

when compared to Fe65 (

Borg et al., 1998; Sastre et al.,

1998; Lee et al., 2003

) decreased neprilysin activity in

cells and PS

_{fibroblasts (not shown). Similar}

potenti-ation of neprilysin expression by Fe65 and Tip60 trans-

HEK293 cells (40%, 46%, and 47% of inhibition of

nep-rilysin activity compared to control for X11

α, X11β, and

fection was also observed in AICD-transfected

PS-defi-cient fibroblasts (136% ± 6%, AICDC50+Fe65+Tip60

X11

γ, respectively, n = 5) (

Figure 5

F).

versus AICDC50 alone and 135% ± 6.2%, AICDC59+

Fe65+Tip60 versus AICDC59 alone; p < 0.0005). Inter-

␤APP and APLPs Complement Each Other

to Control Neprilysin In Vitro and In Vivo

estingly, AICDC50 and AICDC59 increase renal

nepri-lysin promoter transactivation in HEK293 cells (not

To test whether the endogenous PS-dependent

prod-uct controlling neprilysin activity was indeed AICD, we

shown), a phenotype further potentiated by Fe65 and

regulator of neprilysin transcription both in vitro and

in vivo. However, it should be noted that the extent of

inhibition of neprilysin activity appeared lower in

βAPP

_{cells than in PS}

_{fibroblasts. Therefore, we}

examined whether neprilysin activity and expression

could be controlled by other

βAPP-like proteins.

Inter-estingly, APLP2-deficiency in fibroblasts triggers a

de-crease in both neprilysin activity and expression that

is similar to the reductions observed in

βAPP

_cells

(

Figures 6

A–6D). Neprilysin activity is fully restored by

APLP2 cDNA transfection in APLP2

_fibroblasts

(125.1 ± 6.9 of control, n = 3; p < 0.05;

Figure 7

A).

How-ever, the absence of both

βAPP and APLP2 in

fibro-blasts resulted in an even more dramatic reduction in

neprilysin activity and expression (80% ± 2.6%

reduc-tion of activity in homogenate, n = 8; p < 0.0001;

Figure

6

A; 87% ± 2.% reduction of activity on intact cells, n =

6; p < 0.0001;

Figure 6

B; and 92% ± 0.7% reduction of

expression in APP

_APLP2

_{versus control}

fibro-blasts, n = 3; p < 0.0001;

Figures 6

C and 6D), while

ECE-like and IDE-like immunoreactivities remained

un-affected (not shown). Importantly, double

βAPP/APLP2

deficiency also led to decreased NEP mRNA

expres-sion (30% reduction in two independent experiments,

Figure 6

B [inset]). Of most interest is our observation

that cotransfection of Tip60 and Fe65 cDNAs

drasti-cally increase neprilysin activity in wild-type fibroblasts,

but not in APP

_APLP2

_{doubly deficient fibroblasts}

(

Figure 7

B). However, AICDC50 still potentiates

trans-activation of rNEPP1 and rNEPP2 promoters in APP

APLP2

_{fibroblasts (}

_{Figure 7}

_C).

Figure 6. NEP Expression and Activity Are Affected by APP, APLP1,

This apparently synergistic effect led us to

_{size that APP and APLP2, which have homologous C}

termini, could partially complement each other for the

_{control of neprilysin expression. However, it should be}

Experi-noted that the extent of inhibition of neprilysin activity

mental Proceduresin the indicated single or multiple KO fibroblasts

in brain (52% ± 9.8% in APP

_APLP2

_{versus control,}

(A–D) or in mice (E and F) or Alzheimer’s (G–I) brain tissues. (G) NEP

n = 4; p < 0.05;

Figure 6

F) was lower than that observed

of NEP and IDE (I) in L235P-PS1 and F386S-PS1 cases (FAD), two

in the corresponding APP

_APLP2

_{double knockout}

sporadic cases (AD), and control brains (CT). Bars in (B), (F), and

_{fibroblasts. This could be due to another protein that}

_{would complement APP and APLP2 function in brain}

but not in fibroblasts. In this context, it is noteworthy

corre-that, unlike in the brain, fibroblasts totally lack the

βAPP

repre-family member APLP1 (not shown). Therefore we

ined whether the

γ-secretase-derived fragments of

APLP1 (ALID1) and APLP2 (ALID2) could complement

neprilysin activity in APP

_APLP2

_fibroblasts.

In-examined neprilysin activity and expression in

βAPP-

_{deed, both ALID1 and ALID2 significantly increase}

nep-deficient fibroblasts.

βAPP

_{fibroblasts exhibit a}

simi-rilysin activity in APP

_APLP2

_{fibroblasts (142.1 ± 8,}

larly significant reduction of neprilysin activity (30% ±

_{n = 3; p < 0.005 for ALID1 and 138.8, n = 3; p < 0.005 for}

7.6% [n = 7; p < 0.005;

Figure 6

A] and 31% ± 6.7% [n =

_{ALID2, versus control;}

_{Figure 7}

_{D). The fact that APP}

_,

3; p < 0.001;

Figure 6

B] reduction in homogenates and

_APP

_APLP1

_{, and APP}

_APLP2

_{brains all display}

intact cells, respectively, versus control activity) that

_{similar reductions in neprilysin activity (}

_{Figure 6}

_F)

indi-fully matched the reduction in NEP expression (26.1% ±

_{cate that all the members of the APP family control}

ce-6.9% reduction, n = 3; p < 0.05;

Figures 6

C and 6D).

_{rebral neprilysin transcription in vivo. It should be}

Neprilysin activity is fully restored by

βAPP cDNA trans-

_{noted, however, that the mice brains devoid of APP,}

fection in APP

_{fibroblasts (125.6 ± 6 of control, n =}

APLP1, and APLP2 do not exhibit enhanced neprilysin

4; p < 0.01;

Figure 7

A). It is of interest that loss of

βAPP

decrease when compared to double KO brains (not

expression in APP

_{mouse brain triggers a significant}

shown). This suggests that, besides

AICD/ALID-regu-reduction in neprilysin activity (47% ± 3.5% in APP

_{lated neprilysin expression, there exists also a}

constitu-versus control, n = 4; p < 0.005;

Figures 6

E and 6F). This

tive APP/APLP-independent cerebral NEP activity.

suggests that derivatives of

βAPP might also control

In order to establish whether the control of neprilysin

cerebral neprilysin in vivo and supports the notion that

activity was restricted to the APP-related ICDs, we next

a presenilin-dependent,

γ-secretase-mediated cleav-

examined the putative effect of other PS-dependent

γ-secretase-mediated products. Thus, NICD is the

Neuron 548

Figure 7. Neprilysin Activity Is Modulated by ALID1 and ALID2 but Not by NICD or E-Cad/CTF2

APP−/−_{(A), APLP2}−/−_{(A), or APP}−/−_APLP2−/−_{(B–D) fibroblasts were transfected with empty pcDNA3 vector or indicated cDNAs; then NEP}

activity (A, B, and D) or transactivation of rNEPP1 and rNEPP2 promoters (C) were monitored as described in theExperimental Procedures. Error bars in (A), (B), and (D) represent the mean ± SEM of three to four independent experiments. *p < 0.05; **p < 0.005. (E–G) The indicated fibroblasts were transiently cotransfected with 4XCBF-luciferase andβ-gal cDNAs in combination with either empty vector (Ct) or cDNAs encoding myc-tagged m_{⌬ENotch or myc-tagged NICD, and then m⌬ENotch and NICD (E) or NEP (F) expressions were assayed by Western} blot in the indicated cell lines, and luciferase (E) or NEP (G) activities were measured as described in theExperimental Procedures. Bars in (E) and (G) represent the mean ± SEM of three to four independent experiments. NICD expression was also estimated by immunohistochem-istry in PS+/+_{and PS-deficient fibroblasts (H). Note a clear dense label of NICD in the nucleus. (I) E-Cad/CTF2 does not modulate NEP}

expression and activity in HEK293 cells. Bars in (I) represent the mean ± SEM of seven independent experiments.

tracellular fragment liberated from Notch upon

γ-secre-

activity in PS

_{cells. Taken together, these data}

indi-cate that the complementation of neprilysin function by

tase cleavage. To test whether NICD might also affect

neprilysin activity, we created fibroblast- and blasto-

γ-secretase-derived products is specifically elicited by

ICDs of the

βAPP family members.

cyst-derived cell lines transiently expressing the

4XCBF-luciferase reporter gene. In agreement with previously

published works (

Herreman et al., 2000; Zhang et al.,

Neprilysin Expression and Activity Are Increased

in Alzheimer’s Brains of Genetic Origin

2000

), the transient cotransfection of m

⌬ENotch (which

generates NICD upon

γ-secretase cleavage) led to a ro-

We have examined the neprilysin activity of control

hu-man brains and compared them to brain samples of

bust transcriptional activation in wild-type fibroblasts

(

Figure 7

E) and blastocyst-derived cells (not shown),

sporadic or genetic AD origin. Neprilysin activity (

Fig-ures 6

G and 6H) and expression (

Figure 6

I) were not

but not in the equivalent PS-deficient cells (

Figure 7

E).

However, transfection of NICD itself into wild-type and

statistically different between control (CT) and sporadic

AD brains, while Familial Alzheimer’s disease (FAD)

PS-deficient cells induced NICD protein expression

(

Figure 7

E), favored nuclear localization of NICD (

Figure

brains harboring PS1 mutations displayed higher

activ-ity (

Figures 6

G and 6H) and expression (

Figure 6

I).

Inter-7

H), and increased luciferase activity (

Figure 7

E). These

control experiments therefore established that NICD

estingly, insulin-degrading enzyme (IDE) was not

al-tered in FAD brains (

Figure 6

I).

was indeed functionally expressed in our experimental

system. However, regardless of the cell system used,

m

⌬ENotch and NICD were unable to restore neprilysin

Discussion

expression (

Figure 7

F) or neprilysin activity (

Figure 7

G)

in PS-deficient cells. Furthermore, the

γ-secretase-

Several lines of evidence strongly suggest that

nepri-lysin (

Hauss-Wegrzyniak and Wenk, 2002; Hama et al.,

derived C-terminal products of E-Cadherin (E-Cad/

CTF2,

Figure 7

I) (

Marambaud et al., 2002

) and N-Cad-

2001; Marr et al., 2003; Leissring et al., 2003; Iwata et

al., 2001

), endothelin-converting enzyme (ECE;

Eckman

herin (N-Cad/CTF2, not shown) (

Marambaud et al.,

2003

) were also unable to affect neprilysin expression

et al., 2003

), and insulin-degrading enzyme (IDE;

Farris

et al., 2003

) could participate in the catabolism of A

β

and activity. Thus, unlike AICDs and ALIDs, NICD and

expression, and enzymatic activity are dramatically re-

drastically reduces both A

β production and Notch

sig-naling (

De Strooper et al., 1998

). We hypothesize that in

duced in non-neuronal and neuronal cells when they

are devoid of PS1 and PS2 and that this phenotype can

PS1

_{fibroblasts and brain tissue, APLP1 and APLP2}

could undergo

γ-secretase-like cleavage by residual

be mimicked by

γ-secretase inhibitors. We have shown

that the presenilins control NEP activity at a transcrip-

endogenous PS2, thus complementing AICD function,

and thereby control neprilysin expression and activity.

tional level and that PS deficiency does not affect the

expression of IDE, ECE, or other widely distributed in-

Because APLPs lack the A

β sequence (although Aβ-like

peptides seem to be generated from APLP2), their

PS2-tracellular (calpain and proteasome) or ectopeptidase

(aminopeptidase M) activities. We have also shown that

dependent cleavage would not compensate for A

β

re-duction that is triggered by PS1 deficiency. In this

neprilysin expression and activity are significantly

re-duced in brain tissue from mice lacking PS1 and PS2

context, the resulting phenotype would be a reduction

in A

β production but with unaltered or weakly modified

(

Saura et al., 2004

), indicating that the presenilins also

control cerebral neprilysin transcription, expression,

neprilysin levels. Alternatively, we hypothesize that

there is likely to be both a constitutive

(AICD-indepen-and activity in vivo. This highly specific (AICD-indepen-and

physiologi-cal relationship between the presenilins and neprilysin

dent) basal expression of NEP and an inducible

(AICD-dependent) expression of NEP, as suggested by

resid-strongly argues for a major evolutionary and functional

role of neprilysin in the physiological catabolism of A

β,

ual NEP activity in triple KO (APP

_APLP1

_APLP2

₎

mice brains (see

Results

). In PS1

_{fibroblasts and in}

but does not preclude a role for other enzymes in A

β

catabolism.

PS1

_{brain tissue, this constitutive level of expression,}

together with the cumulative effects of residual AICD

The molecular mechanisms by which the presenilins

regulate neprilysin transcription would appear, from the

and ALIDs generated from APP, APLP1, and APLP2 by

the PS2

γ-secretase activity, could be sufficient to

data presented here, to involve the C-terminal

intracel-lular domains of APP and APLP proteins (AICDs and

maintain neprilysin expression and activity in PS1

cells. In contrast, for Notch, there is both absence of

ALIDs). Thus, we have clearly established that

defi-ciency of

βAPP, APLP1, or APLP2 drastically reduces

constitutive basal expression of Notch-targeted genes

and a lack of redundancy in alternate signaling

mole-neprilysin expression and activity in both fibroblasts

and in brain tissues. We have specifically shown that

cules. Another possibility could be that NEP

tran-scription is preserved in PS1

_{cells because APLPs}

A

β42 and the γ-secretase-derived products of Notch,

E-cadherins, and N-cadherins do not support this activ-

γ-secretase cleavage is not affected to the same extent

as APP by PS1 deficiency. However, to our knowledge,

ity. Furthermore, the Fe65 adaptor protein (which binds

to the GYENPTY motif in the cytoplasmic tail of

βAPP/

there is currently no data to support the notion that

PS1 and PS2 have differential/preferential effects on

AICD [

King and Turner, 2004

]) and Tip60 clearly

potenti-ate the AICD-induced upregulation of neprilysin in wild-

γ-secretase cleavage of APP, APLP1, and APLP2.

The experiments described here do more than simply

type fibroblasts but not in PS

_{and APP}

_/APLP2

cells.

confirm the previously and widely held suspicion that

AICD, like NICD, might act as a signaling molecule

in-It has been previously reported that Fe65 and Tip60

stabilize AICD and favor its translocation to the nucleus

volved in transcriptional activation. The experiments

here depict an elegant and unusual mechanism by

(

Kimberly et al., 2001; Kinoshita et al., 2002

). Our

exper-iments have shown that AICD translocates to the nu-

which A

β levels are controlled. Thus, βAPP is

proteolyti-cally processed by presenilin-dependent

γ-secretase,

cleus in the presence of Fe65 and Tip60 and

transacti-vates neuronal and renal neprilysin promoters in TSM1

which generates two distinct types of catabolites, A

β

and AICD (

Figure 8

). One of these products, AICD, then

neurons, HEK293 cells, and APP

_/APLP2

fibro-blasts, respectively. Furthermore, Supergel shift analy-

controls the lifetime of the other product, A

β, by

selec-tively activating the transcription of an enzyme

(nepri-sis indicates that AICDC59 interacts physically with the

neprilysin promoter in the presence of Fe65 and Tip60.

lysin) capable of degrading that other product. Thus,

γ-secretase activity directly controls Aβ production and

Interestingly, X11, which clearly reduces

γ-secretase

cleavage of

βAPP, also drastically reduces neprilysin

then indirectly modulates its degradation. To our

knowl-edge, a similar “self-contained” mechanism for

regulat-activity. Therefore, Fe65 and X11, which trigger

oppo-site effects on A

β production, also elicit an opposite

ing the degradation of enzyme products has not been

described previously; (NB: this does not preclude

phenotype in neprilysin regulation.

Altogether, the similar decrease in neprilysin activity

AICDs from also activating other genes).

If A

β production and degradation are tightly linked,

and expression triggered by the absence of PS or APP/

APLPs, the analogous absence of control of neprilysin

this raises the question of why A

β accumulates in AD.

The net accumulation of A

β in AD pathology likely

re-by Fe65 and Tip60 in these invalidated fibroblasts, and

the opposite phenotype observed with Fe65 and X11

flects the cumulative effect of multiple events acting on

production, fibrillogenesis, and degradation. In many

all lead to the firm suggestion that the endogenous

γ-secretase-dependent fragments controlling neprilysin

forms of AD, especially the late-onset sporadic forms,

it has not been shown that there is increased

β- and

are indeed AICD/ALIDs.

The redundant role of PS1 and PS2 in regulating nep-

γ-secretase activity. In fact, some have suggested that

these forms may reflect defective degradation of A

β

rilysin transcription contrasts with their overlapping but

not redundant roles in modulating A

β peptide and

(

Iwata et al., 2001; Leissring et al., 2003

). Therefore,

AICD levels are likely to be unchanged in these

late-Notch signaling. Thus, PS2 deficiency alone does not

affect

γ-secretase-mediated production of Aβ or Notch

onset forms of AD, and as a result, the AICD-mediated

ability to upregulate neprilysin activity would not be

ef-in vivo (

Herreman et al., 1999

). However, PS1 deficiency

Neuron 550

Figure 8. Model for PS-Dependent Transcriptional Activation of NEP by theβAPP-Intracellular Domain

βAPP undergoes proteolysis by various secretases. Aβ peptide is released by the sequential cleavages triggered by β- and γ-secretases in

the intracellular compartment from which it can be secreted. Once secreted, A_{β is degraded by the ectopeptidase named neprilysin (NEP), a} type II integral protein with catalytic domain facing the extracellular space. Presenilin-dependentγ-secretase cleavage is triggered by a multiproteic complex composed of presenilin 1 (PS1) or presenilin 2 (PS2), nicastrin (NCT), Aph1, and Pen2._{γ-secretase contributes to an} additional cleavage, referred to as⑀ cleavage, that takes place slightly downstream from the γ-secretase site. The action at γ and ⑀ sites gives rise to AICDC59 and AICDC50, respectively. AICDs interact with Fe65 and Tip60 to form an active complex activating NEP mRNA transcription. Increased NEP activity, in turn, leads to increased Aβ degradation.

ficiently brought into play to protect the brain. In con-

AICD. Upregulation of neprilysin by transgenic

overex-pression, at least to modest levels, appears to be

suffi-trast, in those cases of AD arising from mutations in

APP and PS1, which activate

γ-secretase and AICD

cient to reduce brain A

β levels and to pose few toxic

side effects (

Leissring et al., 2003

). This strategy would

production, the principal effect is to produce longer A

β

isoforms such as A

β42. However, although Aβ40 is effi-

also circumvent the other side effects of

γ-secretase

inhibitors, including the potentially self-defeating effect

ciently degraded by NEP, A

β42 is degraded by NEP

both in vitro and in vivo at a 6-fold lower rate, (

Shirotani

of reducing AICD and thus preventing NEP-mediated

degradation of A

β.

et al., 2001

). As a result, the upregulation of AICD (and

thus, NEP expression), which would be anticipated in

subjects with presenilin mutations, would not

com-Experimental Procedures

pletely abolish the accumulation of A

β42 in these

cases. It should be noted that, in agreement with the

above hypothesis, neprilysin expression and activity

were higher only in brain tissues with familial

Alzheim-p97Fe65-deficient fibroblasts were obtained and cultured as

pre-er’s disease linked to various presenilin-1 mutations,

viously described (Vincent et al., 1996; De Strooper et al., 1999;

while sporadic AD cases displayed neprilysin levels

Zhang et al., 2000; Herreman et al., 2000 Armogida et al., 2001;

similar to those exhibited by normal brain tissues (

Fig-

fibro-ures 6

G–6I). Interestingly, PS1 mutations selectively af-

fect neprilysin and do not alter insulin-degrading en-

zyme expression.

cultured in DMEM/10% fetal calf serum containing 2 mM glutamine

The above observations are also of direct practical

and 50_{␮M β-mercaptoethanol. Transient transfections were carried}

interest because they indicate the possibility of new

av-out with DAC 30 (Eurogentec).

enues for controlling A

β levels without directly affecting

γ-secretase. This latter concept is important because of

the various developmental and postnatal side-effects

NEP activity was measured on intact cells or in cell homogenates

associated with the inhibition of

γ-secretase-mediated

with Suc-Ala-Ala-Phe-7AMC in the absence or presence of

phos-cleavage of other signaling molecules, including Notch

phoramidon as described previously (Checler, 1993). When NEP

(

Sisodia and St. George-Hyslop, 2002; Haass and De

activity was measured in brain tissues, brains were homogenized

Strooper, 1999

). Our work now suggests that A

β levels

resus-might be modulated by directly increasing neprilysin

expression, using AICD or small molecule mimics of