Báo cáo khoa học: Shedding of the amyloid precursor protein-like protein APLP2 by disintegrin-metalloproteinases Retinoic acid-induced upregulation of substrate and proteinase ADAM10 during neuronal cell differentiation ppt

Reti-noic acid treatment of two neuroblastoma cell lines upregulated the expres-sion of both APLP2 and ADAM10, thus leading to an increased release of soluble APLP2.. Abbreviations ADAM,

APLP2 by disintegrin-metalloproteinases

Retinoic acid-induced upregulation of substrate and proteinase

ADAM10 during neuronal cell differentiation

Kristina Endres1, Rolf Postina1, Anja Schroeder2, Ulrike Mueller3and Falk Fahrenholz1

1 Institute of Biochemistry, Johannes Gutenberg-University Mainz, Germany

2 ZVTE, Johannes Gutenberg-University Mainz, Germany

3 Institute for Pharmacia and Molecular Biotechnology, University of Heidelberg, Germany

The amyloid precursor protein (APP) is a member of a

protein family in mammals that includes the APP-like

proteins APLP1 and APLP2 [1] All APP⁄ APLP

fam-ily members are type I integral membrane proteins

with large extracellular ectodomains and short

cyto-plasmic tails Compared with APP, both APLPs are

highly homologous in their amino acid sequence (e.g

APLP2⁄ APP 52% identical, 71% similar) [2] and are

proteolytically processed in a similar way The

N-ter-minal ectodomains are released by a shedding enzyme

[2,3], whereas the C-termini remain in the membrane

[2,4,5] and can be further processed to release a cyto-plasmic fragment with signaling properties [4,6,7] Further elucidation of APLP2-processing is of rele-vance with regard to the outstanding function of this protein, which was derived from knockout experiments Whereas a double knockout of APP and APLP1 did not show severe phenotypic changes in mice, the com-bined knockout of APLP2 with both of the other APP family members resulted in postnatal lethality [8,9] This shows that APLP2 and⁄ or one of its proteolytic fragments are essential for normal development and

Keywords

ADAM10; Alzheimer’s disease; amyloid

precursor protein-like protein 2; retinoic acid;

tumor necrosis factor-a converting enzyme

Correspondence

F Fahrenholz, Institute of Biochemistry,

Johannes Gutenberg-University, Becherweg

30, D-55128 Mainz, Germany

E-mail: bio.chemie@uni-mainz.de

(Received 27 June 2005, revised 14

September 2005, accepted 16 September

2005)

doi:10.1111/j.1742-4658.2005.04976.x

Cleavage of the amyloid precursor protein (APP) within the amyloid-beta (Ab) sequence by the a-secretase prevents the formation of toxic Ab pep-tides It has been shown that the disintegrin-metalloproteinases ADAM10 and TACE (ADAM17) act as a-secretases and stimulate the generation of

a soluble neuroprotective fragment of APP, APPsa Here we demonstrate that the related APP-like protein 2 (APLP2), which has been shown to be essential for development and survival of mice, is also a substrate for both proteinases Overexpression of either ADAM10 or TACE in HEK293 cells increased the release of neurotrophic soluble APLP2 severalfold The strongest inhibition of APLP2 shedding in neuroblastoma cells was observed with an ADAM10-preferring inhibitor Transgenic mice with neu-ron-specific overexpression of ADAM10 showed significantly increased lev-els of soluble APLP2 and its C-terminal fragments To elucidate a possible regulatory mechanism of APLP2 shedding in the neuronal context, we examined retinoic acid-induced differentiation of neuroblastoma cells Reti-noic acid treatment of two neuroblastoma cell lines upregulated the expres-sion of both APLP2 and ADAM10, thus leading to an increased release of soluble APLP2

Abbreviations

ADAM, a disintegrin and metalloproteinase; ADAM10DN, catalytically inactive dominant negative mutant form of ADAM10; APLP1, APP-like protein 1; APLP2, APP-like protein 2; APLP2s, cleaved soluble APLP2; APP, amyloid precursor protein; BACE, b-site APP-cleaving enzyme; CS-GAG, chondroitin sulfate glycosaminoglycan; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; PVDF, poly(vinylidene difluoride); RA, retinoic acid; TACE, tumor necrosis factor-a converting enzyme.

survival, and may compensate the lack of either APP or

APLP1 Whereas APP orthologs have been identified in

lower and higher vertebrates, a recent publication

revealed the existence of the first nonmammalian

APLP2 in Xenopus laevis and its overall high

percent-age of conserved amino acids implies an important role

for this member of the APP superfamily [10] Although

BACE [11–13] and the c-secretase [6,22] have

previ-ously been identified as proteinases involved in the

pro-teolytic processing of the APP relatives, it remains to

be shown whether APLPs are also subject to cleavage

by disintegrin-metalloproteinases (ADAMs) which act

as a-secretases for APP [14–16]

Shedding of APLP2 can be induced by activation

of protein kinase C (PKC) in human corneal epithelial

cells [17] Moreover, a decline in the

membrane-anchored C-terminal fragments of APLP1 and APLP2

by the hydroxamic acid-based inhibitors batimastat

and TAPI-2 was shown recently [11] Using deletion

mutants, metalloproteinase-dependent cleavage of

APLPs was shown to occur at a similar distance to the

membrane as is known for APP Thus, an

a-secretase-like activity seems to release the APLP2 ectodomain,

but the proteinases involved are not yet identified

Three members of the ADAM family have been

shown to act as a-secretases [14,15,18] We restricted

our investigations on APLP2 shedding to ADAM10

and tumor necrosis factor-a converting enzyme

(TACE, ADAM17), because purified ADAM9 failed

to cleave a synthetic APP peptide at the major

a-secre-tase cleavage site [19], and ADAM9 knockout mice

exhibit unchanged APP processing [20] ADAM10, in

contrast, was recently shown to process APP in vivo

and to prevent plaque formation in an Alzheimer’s

dis-ease mouse model [16]

ADAM10 and TACE, which cleave APP, have

been implicated in ectodomain shedding of other

sub-strates such as cytokines [21], growth factors and

their receptors [22,23], and adhesion molecules [24]

If ADAMs have several cellular substrates, how are

physiologically relevant processing events

coordina-ted? One possibility is a common up- or

downregula-tion of substrate and sheddase during cell-fate

decisions Differentiation of neuronal cell types

through retinoic acid (RA) leads to the upregulation

of both APP [25] and APLP2 [26,27] Therefore, we

investigated the effect of RA on ADAM10 and

TACE expression in neuroblastoma cell lines In this

study we provide evidence for a common

upregula-tion of ADAM10 and its newly identified substrate

APLP2 by RA-induced neuronal cell differentiation

which resulted in an enhanced release of neurotrophic

secreted APLP2 [28]

Results Phorbol-12-myristate-13-acetate-induced APLP2 ectodomain shedding

To study the effect of the PKC activator phorbol-12-myristate-13-acetate (PMA) on endogenous APLP2

SH-SY5Y cells with 1 lm PMA and performed Western blot analysis of proteins from cell supernatants It has been shown that a large fraction of APLP2 and its secre-ted soluble derivative is modified by the addition of chondroitin sulfate glycosaminoglycan (CS-GAG) at a single site (Ser614) in the extracellular domain This gives rise to the secretion of molecules with an apparent molecular mass between 130 and 170 kDa (Fig 1) Two minor sharp bands between 95 and 120 kDa probably represent, according to earlier studies, APLP2s and truncated APLP2s without CS-GAG-modification (for post-translational modification of APLP2 see Slunt

et al [2] and Thinakaran and colleagues [29,30]) PMA treatment of all tested cell lines resulted in a significant increase in secreted endogenous APLP2 indicating that shedding of APLP2, like that of APP, is stimulated by PMA in neuronal and non-neuronal cell lines (Fig 1)

Inhibition of APLP2 ectodomain shedding

by metalloproteinase inhibitors

It is known that the shedding of various transmem-brane substrates is inhibited by hydroxamic acid-based inhibitors [31,23,32] GM6001, a broad-spectrum hydroxamic acid-based inhibitor of matrixmetallopro-teinases (MMPs) and ADAMs, decreased basal APPsa and APLP2s secretion to 60 and 75%, respectively, of untreated cells (Fig 2A,B, lanes 1 and 3) and reduced the PMA-stimulated amount of both shed ectodomains

to almost the level of control cells without inhibitor

-PMA

98 kDa

148 kDa

Fig 1 Enhancement of APLP2 secretion in HEK293, SKNnc and SH-SY5Y cells by the PKC activator PMA PMA was added at a final concentration of 1 l M for 4.5 h, proteins in the cell supernatants were then precipitated and analyzed by western blotting using the antibody D2II A representative example of three independent experi-ments is shown Arrows indicate differentially modified APLP2s.

(Fig 2A,B, lanes 2 and 4) This suggested participation

of either ADAMs and⁄ or MMPs in the processing of

APLP2 There was more pronounced inhibition of

constitutive shedding by the inhibitor GI254023X,

which has a 100-fold higher potency to inhibit

recom-binant ADAM10 than recomrecom-binant TACE [33,34]

When compared with solvent-treated control cells,

both APPsa and APLP2s were decreased to 30%

(Fig 2A,B, lanes 5 and 6), showing that ADAM10 is

strongly involved in the shedding of APLP2

With both inhibitors the amount of full-length

APLP2 was comparable with control cells (Fig 2A)

and did not increase upon inhibited processing

Because a-secretase cleavage of APP occurs at the

surface of neuronal cells [35], only a small fraction of

the total cellular APP is cleaved, which generally does not result in a decrease in the full-length protein [36,37] Therefore, reduction of APLP2 proteolysis

by hydroxamic acid-based inhibitors might also affect only minor pools of the cellular protein resulting in an unchanged steady-state level

To compare the cell-based inhibitory effect of GI254023X on APLP2 shedding with recently pub-lished data for shedding of other ADAM substrates like the interleukin-6 receptor [38], we applied the inhibitor in concentrations ranging from 0.3 to 10 lm

to SH-SY5Y cells (Fig 2E) The IC50value for inhibi-tion of APLP2 shedding by GI254023X was in the micromolar range (1.7 lm) showing a reduction of potency in cellular assays as compared to its effect on

APLP2s

A

E

B

APLP2FI

APPs α

98 kDa

98 kDa

200 150 100 50 0

150

100

50

-+

+ + GM

125

100

75

50

10

0

log c [M]

GI254023X 0 0,3 0,6 1,3 2,5 5 10 µM

98 kDa

PMA GM

0

APLP2 secretion in % of control

98 kDa

Fig 2 Influence of metalloproteinase inhibi-tors on APPsa and APLP2s secretion of neuro-blastoma cells Detection of shed (A) APLP2 and (B) APPsa in SKNMC cells treated with metalloproteinase inhibitors GI254023X, GM6001 and the inactive GM6001NK were added at a final concentration of 10 l M for overnight preincubation, proteins of the cell supernatant were then collected for 4.5 h PMA (1 l M ) was added directly during the collection period Secreted APLP2s and APPsa were detected as described in Experi-mental procedures (lane 1, control; lane 2, PMA; lane 3, GM6001; lane 4, PMA ⁄ G6001; lane 5, control; lane 6, GI254023X) Full-length APLP2 (APLP2Fl) was analyzed in cell lysates (A, lower) to confirm that the inhibi-tors did not alter steady-state levels of the protein Representative blots are shown Quantitative analysis of (C) APLP2 and (D) APPsa secretion Quantification for APLP2 was carried out taking into account all three APLP2 protein forms Values are the mean ± SD of three independent experi-ments Control cells treated with the solvent

or the inactive compound GM6001NK (indi-cated as GM –) were set to 100% (One-way ANOVA: *P < 0.05, **P < 0.01) (E) Detec-tion of constitutive APLP2 shedding in SH-SY5Y cells Cells were pretreated with increasing doses of GI254023X (0.3–10 l M ) for 30 min After 4 h treatment with freshly added inhibitor, the conditioned media were harvested and the amount of secreted APLP2 was determined Data represent the mean ± SD of three independent experi-ments performed in duplicate The inhibitor dose–response curve was generated using the software GRAPHPAD PRISM 4.02 (GraphPad Software Inc., San Diego, CA, USA).

recombinant ADAM10 with IC50 values in the

nano-molar range [33] In comparison, inhibition of the

interleukin-6 receptor shedding in COS cells occurred

with a potency of 1.8 lm [38] and therefore was in the

same range as found for cellular APLP2 shedding

Inhibition of APLP2 ectodomain shedding by a

specific b-secretase inhibitor

Another proteinase suggested to be an APLP2-cleaving

enzyme is BACE-1 [11,13] To elucidate whether, in

cells of neuronal origin, APLP2 is processed by

b-secre-tase, we tested the effect of the tripeptidic b-secretase

inhibitor [(N-benzyloxycarbonyl-val-leu-leu-leucinal)

Z-VLL-CHO] on APLP2 shedding in the human

astro-glioma cells U373 These cells overexpress human

wild-type APP and therefore allow detection of

BACE-1-generated secreted APPsb, which is normally

found at very low concentrations in the cell supernatant

[41] As shown in Fig 3, both ectodomains were

reduced significantly by applying the b-secretase-specific

inhibitor For APPsb we found a decreased shedding of

50% of control cells For APLP2s shedding was

inhibited to a significant but lesser extent (reduction of

 30% compared with control cells)

Because the antibody available against the APLP2 extracellular region (D2II) recognizes both the BACE-1- and a-like cleavage product of APLP2, APLP2s in cell supernatants reflect the effect of both shedding pro-cesses Probably therefore the effects on the a-like clea-vage of APLP2 by metalloproteinase inhibitors (Fig 2)

or on the b-like cleavage by a BACE-1 inhibitor (Fig 3) are probably not as strong as for the processing of APP, which is monitored by specific antibodies (a-cleavage, 6E10, Fig 2B; b-cleavage 192 Wt, Fig 3A)

Enhancement of APLP2 secretion by over-expression of the a-secretases ADAM10 and TACE

To identify the proteinases that participate in APLP2 shedding, we examined cells overexpressing the a-secret-ase ADAM10 or TACE (Fig 4A–C) Stable overexpres-sion of either proteinase resulted in
2.5–3.5-fold more soluble APLP2 in the culture supernatant than in con-trol cells (Fig 4A,B) Because expression levels of the two proteinases differed (TACE being expressed at higher levels, Fig 4C), we are not able to determine from the data which of the two enzymes preferentially cleaves APLP2 In all cases, overexpression did not sig-nificantly alter the steady-state levels of cellular APLP2 (data not shown), therefore the observed effects are not due to enhanced expression levels of APLP2

Effect of a dominant negative mutant of ADAM10

on APLP2 shedding

To verify the APLP2-shedding activity of endogenous ADAM10, we used a cell line with stable overexpres-sion of a dominant negative form of ADAM10 (Fig 4F) This mutant protein carries the E384A point mutation in the zinc-binding region of ADAM10, which is known in Drosophila melanogaster [40] and in HEK293 cells [14] to suppress endogenous ADAM10 activity HEK ADAM10DN cells showed a decreased APLP2 secretion of
60% compared with nontrans-fected HEK293 cells (Fig 4D,E), whereas expression

of full-length APLP2 was not significantly affected (data not shown) Thus, dominant negative ADAM10 inhibits the endogenous APLP2 sheddase activity

Influence of overexpressed ADAM10 on the proteolytical processing of APLP2 in transgenic mice

Cleavage of APLP2 in vivo was demonstrated by west-ern blots comparing brain homogenates from FVB⁄ N

APPs b APLP2s control

98 kDa

A

B

100

50

0

*

*

β-secretase-Inhibitor II

98 kDa

148 kDa

β-secretase-Inhibitor II

Fig 3 Effect of the b-secretase inhibitor II on the ectodomain

shedding of APLP2 in astroglioma cells overexpressing APP (A)

Western blots of secreted APPsb and APLP2s upon b-secretase

inhibitor treatment of U373hwtAPP cells Following preincubation

for 18 h with 25 l M of the b-secretase inhibitor II, shedding of

APPsb and APLP2s was analyzed in western blots with the

anti-bodies 192 Wt or D2II (B) Quantitation of APPsb and APLP2s The

amount of shed proteins was quantified in three independent

experiments For secreted APLP2 all detectable protein bands

above the 98 kDa marker band were taken into account (unpaired

Student’s t-test: *P < 0.05).

mice and APLP2 knockout mice (Fig 5A) In FVB⁄ N

mice (Wt) the antibody D2II against the N-terminal

part of APLP2 detected a double band (Fig 5A, lane

1) The CS-GAG-modified protein species were almost

not detectable according to the low levels of this form

in the brain as described for rat neuronal tissue [41]

By using antibody CT12, two C-terminal processing

products of APLP2 were identified (C-stub I and II,

Fig 5A, lane 1) These stubs were also detected in

HEK cells which had been treated for 20 h with the

c-secretase inhibitor DAPT before cell lysis (results not

shown)

To examine the a-like cleavage of APLP2 by ADAM10 in vivo, we investigated the influence of overexpressed ADAM10 in a transgenic mouse line These mice overexpress bovine ADAM10 under the control of a neuron-specific Thy1 promoter [16] Expression of the HA-tagged ADAM10 protein in brains of transgenic mice was verified by immunoblot-ting with the anti-HA serum Y-11 Both the immature and the mature forms of ADAM10 were detectable with a dominance of the catalytically active, mature form (Fig 5B)

To analyze APLP2 processing, soluble and mem-brane-bound proteins from brain homogenates were subjected to immunoblotting using either the D2II or the CT12 antibody We detected an enhanced amount

of secreted APLP2 protein fragments (170%) by com-paring ADAM10 transgenic mice with wild-type litter-mates (Fig 5C,D) When we examined the amount

of C-terminal stubs, we noticed a roughly twofold increase in both C-stubs in ADAM10 transgenic mice (Fig 5C,D) No fragment corresponding to an APLP2 Cb-stub could be detected by immunoblotting with the CT12 antibody in mouse brain homogenates, and therefore both identified C-stubs probably correspond

to a-secretase-like cleavage products

To exclude the possibility that the observed effects result from an altered expression intensity due to over-expression of the proteinase, we performed real-time RT-PCR experiments with mouse brain mRNA APLP2-mRNA levels in transgenic and in control mice were not significantly different (P > 0.4; n ¼ 5, data not shown)

Effect of RA on APP, APLP2 and ADAM10 expression in neuroblastoma cell lines Because APP and APLP2 expression is enhanced during neuronal differentiation [26,27], we wanted to elucidate the effect of RA-induced differentiation of neuroblastoma cell lines on ADAM10 and TACE expression and on the release of secreted APLP2 and APPsa For neuronal (N)-type SH-SY5Y cells, differ-entiation by RA was accompanied by the generation

of long cellular outgrowths Under the same condi-tions, the more Schwann-like SKNMC cells changed their morphology only slightly but revealed strongly decreased proliferative properties (Fig 6A); for a char-acterization of both cell lines during differentiation see Voigt and Zintl [42]

The effect of RA-induced differentiation on either the substrate APLP2 or the proteinase ADAM10 was analyzed using real-time RT-PCR for quantification of mRNAs At the mRNA level, APLP2 was increased

HEK

A

B

E

D

98 kDa

∗∗

∗∗

∗∗

400

125 100 75 50 25 0

300

200

100

0

HEK

control

HEK ADAM10

ADAM10 control ADAM10DN

HEK HEK ADAM10DN HEK

TACE

TACE

98 kDa

64 kDa

64 kDa

AD10 DN T

Fig 4 Influence of ADAM10, TACE and dominant negative

ADAM10, overexpressed in HEK293 cells, on APLP2 shedding (A)

Immunoblot of secreted APLP2 with antibody D2II in ADAM10

and TACE overexpressing cells (B) Quantification of APLP2s in

ADAM10 and TACE overexpressing cells (mean ± SD of three

experiments performed in duplicate, unpaired Student’s t-test:

**P < 0.01) As control, HEK cells transfected with the empty

vec-tor pcDNA3 were used and set to 100% A representative example

is shown (C) Immunoblot of overexpressed ADAM10 and TACE.

The overexpressed proteinases were detected in cell lysates by an

anti-HA serum (D) Immunoblot of secreted APLP2 in ADAM10DN

overexpressing cells A longer exposure time as in (A) was chosen

to demonstrate the reduction of basal secretion of APLP2 by

ADAM10DN (E) Quantification of APLP2s in ADAM10DN

overex-pressing cells (mean ± SD of three experiments performed in

dupli-cates, unpaired Student’s t-test: **P < 0.01) (F) Immunoblot of

overexpressed dominant negative ADAM10 The overexpressed

mutated form of ADAM10 was detected in cell lysates by an

anti-body against the fused Flag-epitope.

significantly in both RA-differentiated cell lines

SH-SY5Y and SKNMC (Fig 6B) Also, ADAM10 mRNA

was strongly increased as we have recently shown for

SH-SY5Y cells [45] Interestingly, both mRNA species were induced more strongly in the N-type neurobla-stoma cell line SH-SY5Y than in the more

Schwann-98 kDa

14 kDa

Wt

A

B

APLP2 KO

APLP2s

C-stub I C-stub II

C-stub I C-stub II

98 kDa

64 kDa

Wt

Wt

AD10

ADAM10

immature mature

98 kDa

14 kDa

APLP2s

APLP2s

**

250

200

150

100

0

50

**

**

C-stubs

Fig 5 Analysis of APLP2 proteolysis in transgenic mice overexpressing ADAM10 (A) APLP2 processing products in mice The high specific-ity of the antibodies D2II and CT12 (recognizing either an N-terminal epitope or an epitope at the very end of the C-terminus) is demonstra-ted by comparing brain homogenates of wild-type with APLP2 knockout mice in western blots (B) Detection of overexpressed ADAM10 in transgenic mice ADAM10 transgenic mice were 10 weeks old As controls we used nontransgenic littermates (Wt) of the same age (C) Detection of APLP2s and the membrane-bound C-stubs The amounts of shed APLP2 and the C-terminal stubs were quantified by Western blotting using membrane and soluble fractions derived from brain homogenates (D) Quantitation of APLP2 processing products in transgenic mice The values of shed APLP2 (APLP2s) and both C-stubs (C-stub I and C-stub II) were quantified for eight animals of each group in at least two independent western blot experiments and normalized to the full-length protein form (mean ± SD, unpaired Student’s t-test:

*P < 0.05, **P < 0.01).

control

control

RA

RA

SH-SY5Y

SH-SY5Y

SKNMC

SKNMC 300

∗

∗∗

∗∗

∗∗

∗∗

200

APLP2 ADAM10 APLP2 ADAM10 BACE

100

0

Fig 6 Morphological changes and mRNA levels in neuroblastoma cell lines upon RA-treatment Cells were treated for 4 days with 1 l M RA (A) Microscopic image of RA-differentiated neuroblastoma cell lines Morphological changes as cellular outgrowths and loss of adherence were determined as markers of differentiation using light microscopy (B) Real-time RT-PCR for mRNA quantitation Changes in mRNA for APLP2 and the proteinases ADAM10 and BACE-1 were investigated using real-time RT-PCR Experiments were performed three times in duplicate and amounts of mRNAs were normalized to GAPDH mRNA Values are given as mean ± SD and results obtained with control cells were set to 100% (unpaired Student’s t-test: *P < 0.05, **P < 0.01).

like SKNMC cells As APLP2 is also known to be

processed by BACE (see above), we also quantified

the mRNA of BACE-1 in SH-SY5Y cells Although

ADAM10 mRNA was induced to
250% compared

with undifferentiated cells, we found only a slight, but

significant increase in the amount of BACE-1 mRNA

(Fig 6B and 147% of control) At the protein level,

the enhancement of APLP2 and ADAM10 was

con-firmed for both cell lines (Fig 7A,B) Again in the

N-type SH-SY5Y the increase in both, the APLP2 and

the ADAM10 protein was stronger than in SKNMC

cells, where significant increase occurred only in the

immature form (Fig 7B)

In contrast to ADAM10 expression, we could not

detect increased TACE protein levels upon RA

treat-ment in our experitreat-ments (Fig 8) Although TACE

remained unchanged in SH-SY5Y cells, the amount of

the pro- and the mature form of this proteinase even

decreased in SKNMC cells, revealing reduced stability

compared with ADAM10 Therefore, the concerted

upregulation of APLP2 and its sheddase during

RA-induced neuronal differentiation appears to be

specific for ADAM10

In both neuroblastoma cell lines we found, upon

RA treatment, an increase of APLP2 shedding Soluble

APLP2 in supernatants of differentiated cells was

enhanced to 150% for SH-SY5Y and 180% for

SKNMC compared with undifferentiated control cells

(Fig 9A) Also, in SH-SY5Y cells the secretion of

APPsa was found to be enhanced significantly to

> 200% of control cells due to increased expression of the a-secretase ADAM10 This phenomenon was also seen in SKNMC cells although to a lesser extent (Fig 9B)

Discussion

We report the cleavage of the mammalian APP-related protein APLP2 by the disintegrin and metalloprotein-ases ADAM10 and TACE (ADAM17), and a common upregulation of ADAM10 and its substrate by RA The main criteria for the involvement of ADAMs, the enhancement of APLP2 shedding by phorbolesters and decreasing amounts of APLP2s by hydroxamic acid derivatives, were fulfilled Overexpression of ADAM10 as well as of TACE resulted in increased secretion of APLP2s from cultured cells Also, a dom-inant negative form of ADAM10 reduced the shedding

of APLP2

GI254023X displayed the most pronounced effect by reducing APLP2s to
30% of control cells, we con-clude that ADAM10, as shown for APP [14], plays an important role in the secretion of the APLP2 ecto-domain We were also able to demonstrate the influence of the a-secretase ADAM10 on APLP2 processing in vivo Transgenic mice with neuronal over-expression of ADAM10 showed significantly increased amounts of shed APLP2 as well as C-terminal process-ing products

SH-SY5Y

SH-SY5Y

SKNMC

SKNMC

98 kDa

98 kDa

64 kDa

control RA

+

-RA

immature mature

control RA

A

B

200

∗∗

∗∗

∗

150

100

50

0

+

200

150

100

50

0

200

150 100 50 0

Fig 7 Expression of APLP2 (A) and ADAM10 (B) in differentiated neuroblastoma cell lines Cell lysates of RA-differentiated SH-SY5Y and SKNMC cells were subjected

to 7.5% SDS ⁄ PAGE, and the proteins were detected by immunoblotting using primary antibodies against the C-termini Experi-ments were performed three times in dupli-cate, representative immunoblots are shown Values are given as mean ± SD and results obtained with control cells were set to 100% (unpaired Student’s t-test:

*P < 0.05, **P < 0.01).

Because soluble APLP2 was shown to induce

neuro-genesis in the subventricular zone of adult mouse brain

[44] and enhances neurite outgrowth [28], the

proteolyt-ical processes that generate APLP2s may be important

for the generation and survival of neuronal cells The

elevation of APP and APLP1 and APLP2 in

differenti-ated SH-SY5Y [27] suggests an important function for

the expression and proteolysis of APP family members

especially in neuronal cell populations In support of

this hypothesis, we found enhanced secretion of the

extracellular domains of APP and APLP2 upon RA

treatment, which might correspond to increased

expres-sion of ADAM10 in both SH-SY5Y and SKNMC cell

lines We cannot completely rule out the possibility

that the increase in secretion of soluble APLP2

follow-ing treatment with RA may also be due to the increase

in the amount of APLP2 and not because of the

increase in ADAM10 expression But because the

BACE-1 mRNA level was increased to a lesser extent,

a major role of the nonamyloidogenic pathway and

ADAM10 in differentiating neuronal cells may be sup-posed Recent findings [43] demonstrate a conserved binding site for retinoid receptors in the promoter sequence of ADAM10 and an increase of promoter activity by RA These results suggest a RA-induced regulation of this disintegrin-metalloproteinase by nuc-lear receptors Because TACE was not positively affec-ted by RA, but even degraded in SKNMC cells, we demonstrate again a higher stability of ADAM10 com-pared with TACE, which was also selectively degraded after PMA treatment of cultured cells [45]

In late-onset Alzheimer’s disease there is genetic, metabolic and dietary evidence for defective retinoid transport and function [46–48] In accordance with these findings, is the observation that the impairment of long-term potentiation induced by experimental vitamin A deficiency in adult mice can be reversed by direct application of RA to hippocampal slices [49] Recently,

we demonstrated that overexpression of ADAM10

in APP[V717I] transgenic mice prevented plaque

immature mature

98 kDa RA 200 150 100 50 0

control RA RA

TACE expression in % of control

200 150 100 50 0

TACE expression in % of control

Fig 8 Expression of TACE in differentiated

neuroblastoma cell lines SH-SY5Y and

SKN-MC cells were differentiated with RA for

4 days, and the mature and the immature

form of the proteinase were detected by

immunoblotting Values are given as mean

± SD of three independent experiments,

and results obtained with control cells were

set to 100% (unpaired Student’s t-test:

*P < 0.05, **P < 0.01).

A

APPsα SKNMC

APLP2s

200

100

0

200 150 100 50 0

Fig 9 Proteolytical processing of APP and APLP2 in RA-treated neuroblastoma cells Western blots and quantification of (A) APLP2s and of (B) APPsa in RA-differentiated neuroblastoma cell lines Cells were treated as described in Experimental procedures Precipitated proteins of cell supernatants were subjected to 7.5% SDS ⁄ PAGE and immunoblotted Detection was performed with the antibodies 6E10 and D2II Values of the quantitative analysis are mean ± SD and significances were determined using paired Student’s t-test (*P < 0.05, **P < 0.01) Experiments were performed three times in duplicate, representative immunoblots are shown.

formation and rescued the impairments of hippocampal

long-term potentiation, thus suggesting a beneficial role

of the a-secretase ADAM10 in memory and learning

[16] Because ADAM10 together with its substrates is

upregulated via RA our results suggest that bioactive

retinoids in the hippocampus could lead to an increased

a-secretase activity and to an increased release of the

neurotrophic-soluble ectodomains of APP and APLP2

Further studies are necessary to support this conclusion

in vivo and to delineate the regulatory mechanism of

RA-induced a-like cleavage of APLP2

Experimental procedures

Materials

PMA and all-trans-RA were purchased from Sigma (St

Louis, MO, USA), the broad-spectrum inhibitor GM6001

(Galardin) and the corresponding inactive control

com-pound (GM6001NK), as well as the b-secretase inhibitor II,

were from Calbiochem (San Diego, CA, USA) Each was

dissolved as stock in dimethylsufoxide and kept at)20
C

Primary antibodies

The following antibodies were used for western blot

analy-sis: D2II, a rabbit polyclonal antibody against the

N-termi-nus of APLP2; CT12, a rabbit polyclonal antibody against

the C-terminus of APLP2 (both kindly provided by

G Thinakaran, University of Chicago, IL); 6E10 (Signet

Laboratories, Dedham, MA, USA) against APPsa; 192 Wt

(S Sinha, Elan Pharmaceuticals, San Francisco, CA, USA)

against APP residues 591–596, detecting only

b-secretase-cleaved soluble APP (APPsb) antibodies against the

C-ter-mini of human ADAM10 and 17 (Chemicon, Temecula,

CA, USA) Overexpressed proteinases were detected with

the anti-HA serum Y-11 (Santa Cruz Biotechnology, Santa

Cruz, CA, USA) or anti-Flag serum M2 (Stratagene, La

Jolla, CA, USA)

Constructs and mutagenesis

The cDNAs of murine TACE [50] and bovine ADAM10 [14]

were fused with a DNA-sequence coding for a hemagglutinin

epitope (YPYDVDDYA), and dominant negative ADAM10

was tagged with a Flag-epitope (DYKDDDDK) as

des-cribed previously [14] Expression of the tagged proteinases

was performed by using the vector pcDNA3 (Invitrogen,

Carlsbad, CA, USA)

Cell culture and transfections

HEK293 cells stably overexpressing either HA-tagged

ADAM10, Flag-tagged dominant negative ADAM10 or

ADAM10DN and HEK TACE, respectively) were cultured

in Dulbecco’s modified Eagle’s medium (DMEM; containing 10% fetal calf serum, 2 mm glutamine, 100 UÆmL)1 penicil-lin, 100 lgÆmL)1streptomycin) SKNMC cells were cultured

in DMEM complete medium supplemented with 1% sodium pyruvate, and SH-SY5Y cells were cultivated in Ham’s F12

glutamine, 100 UÆmL)1 penicillin and 100 lgÆmL)1 strepto-mycin] For the astroglioma cell line U373 MEM supplemen-ted with 10% (v⁄ v) fetal bovine serum, 2 mm glutamine,

100 UÆmL)1penicillin, 100 lgÆmL)1streptomycin, 1% (w⁄ v)

was used

Stable transfections of HEK293 cells were performed by using the calcium phosphate precipitation method followed

by selection of transfected cells with G418 (1 mgÆmL)1) For differentiation of the neuroblastoma cell lines, cells were seeded on 10 cm culture plates after adjusting the cell

cells) and grown for 72 h The medium was replaced by fresh phenol red-free medium containing 1 lm RA, the cells were incubated for 4 days, and the RA-containing medium was changed daily

Western blot analysis of TACE and ADAM10

Cell pellets were washed with NaCl⁄ Pi and dissolved in Laemmli buffer containing 100 mm dithiothreitol, heated to

and transferred to poly(vinylidene difluoride) (PVDF) mem-branes Bound antibodies against the endogenous or over-expressed proteinases were visualized by applying alkaline phosphatase coupled antibodies and the chemiluminescence substrate CDPstar (Tropix, Foster City, CA, USA) Emit-ted light was detecEmit-ted by using a digital camera and quanti-fied with the software aida 3.50 (Raytest, Straubenhardt, Germany)

Western blot analysis of APP, APLP2 and their processing products

Cells were grown close to confluency, washed with serum-free culture medium and incubated for 4.5 h in serum-serum-free

penicillin, 100 mgÆmL)1 streptomycin, 10 lgÆmL)1 fatty acid-free bovine serum albumin and activators or inhibi-tors as indicated PMA (1 lm) was added directly to the serum-free harvesting medium (with 2 mm glutamine,

4.5 h The inhibitors GM6001, its negative control and GI254023X (10 lm) were added to the cells 18 h prior har-vesting and also to the harhar-vesting medium For the dose– response curve of GI254023X SH-SY5Y cells were

pre-incubated for 30 min with varying amounts of the

inhib-itor followed by a harvesting period of 4 h with freshly

added inhibitor Proteins of the culture medium were

pre-cipitated with 10% trichloroacetic acid and collected by

centrifugation The pellets were washed twice with ice-cold

acetone, dried and dissolved in Laemmli buffer containing

Aliquots corresponding to equivalent protein contents of

blotted onto PVDF membranes Soluble APLP2 was

detected with antibody D2II (1 : 2500), followed by

incu-bation with anti-rabbit serum either coupled to alkaline

phosphatase (Tropix) or35S labeled (Amersham Biosciences,

Arlington Heights, IL, USA) Shed APPsa and APPsb

were detected by using the antibodies 6E10 and 192 Wt,

respectively, in combination with secondary antibodies

Bound antibodies were visualized by using a digital camera

or the BAS Reader (Fujifilm, Du¨sseldorf, Germany), and

quantified as described above For detection of full-length

APLP2 and its membrane-bound C-stubs, cells were

centri-fuged for 3 min, 960 g, 4
C An aliquot of the cells was

taken for quantification of the protein content The

Nu-PAGE buffer (Invitrogen) containing 100 mm

dithio-threitol, heated to 70
C for 10 min, separated on 4–12%

Nu-PAGE gels (Invitrogen) and transferred to PVDF

membranes As primary antibody we used CT12 Detection

of APLP2 protein fragments was performed as described

above for the soluble proteins

Preparation of mouse brain homogenates from

transgenic mice

The generation of transgenic mice with neuron-specific

overexpression of bovine ADAM10 has been described

pre-viously [16] Transgenity of mice was confirmed by PCR

ADAM10 proteins by western blotting Mice were chosen

for the experiments with a 1.3-fold increase in the amount

of ADAM10 compared with their wild-type litter-mates

Brains of 10-week-old mice (ADAM10 or wild-type

non-transgenic littermates) were dissected and homogenized in

200 mm Tris⁄ HCl (pH 8.4) in the presence of proteinase

inhibitors (complete mini, Roche, Mannheim, Germany)

Homogenates were centrifuged at 135 000 g for 1.75 h at

4
C for sedimentation of cellular membranes The

superna-tants containing the soluble proteins were removed and the

membrane pellet was suspended in NaCl⁄ Tris The protein

concentrations of both fractions were determined Proteins

were separated on polyacrylamide gels and blotted onto

PVDF membrane as described above As secondary

anti-body we used35S-labeled secondary antibodies For

quanti-fication the BAS Reader (Fujifilm) and the software

aida3.50 were used

Real-time RT-PCR

Total RNA was isolated using the RNeasy Kit (Qiagen, Hil-den, Germany) RNA concentration and quality was deter-mined by spectrophotometry Aliquots of the RNAs were dissolved in RNAse-free water (Sigma) to a concentration of

50 ngÆlL)1 Real-time RT-PCR primers were designed for human GAPDH, ADAM10, BACE and APLP2 from Gene bank mRNA (cDNA) sequences utilizing the primer express1.5 software (Applied Biosystems, Foster City, CA, USA)

AT-3¢, GAPDH_rev 5¢-TCATTGTCGTACCAGGAAAT GAGCTT-3¢; ADAM10_for 5¢-CTGGCCAACCTATTTG TGGAA-3¢, ADAM10_rev 5¢-GACCTTGACTTGGACTG CACTG-3¢; BACE_for 5¢-GTTATCATGGAGGGCTTC TACGTT-3¢, BACE_rev 5¢-GCTGCCGTCCTGAACTCA

CAC-3¢, APLP2_rev 5¢-GGTTCTTGGCTTGAAGTTCT GC-3¢

Real-time RT-PCR was performed using the one-step QuantiTectSYBRGreen RT-PCR-Kit (Qiagen), the ABI-Prism 7000 (Applied Biosystems), 250 ng RNA and the specific primer pairs (0.5 lm of each primer) Reverse

fol-lowed by 45 PCR cycles (one cycle contained the following steps: 15 s at 95
C; 30 s at 55
C; 30 s at 72
C) The spe-cificity of each primer pair was confirmed by melting curve analysis and agarose gel electrophoresis The quantity of mRNA was calculated using either the DDCt method, when PCR efficiency was close to 100%, or a standard curve (e.g for BACE) The mRNA of the housekeeping gene GAPDH was unchanged under differentiation conditions, and all other mRNAs were normalized to it

Acknowledgements

We thank A Roth for excellent technical assistance;

R Black for the murine TACE cDNA; C Prinzen for introduction of the HA-tag into the TACE cDNA, and G Thinakaran for providing the APLP2 cDNA and the antibodies CT12 and D2II We are grateful to

Dr I Hussain, Glaxo SmithKline (Harlow, UK) for putting the inhibitor GI254023X at our disposal This work was supported by the DFG priority program

1085⁄ 3-Cellular mechanisms of Alzheimer’s disease References

1 Coulson EJ, Paliga K, Beyreuther K & Masters CL (2000) What the evolution of the amyloid protein pre-cursor supergene family tells us about its function Neurochem Int 36, 175–184