Studies with b- and c-secretase inhibitors, as well as with the metalloproteinase inhibitor GM6001, revealed an inhibition of neuregulin-1 processing in human astroglioma cell line U373;
Trang 1Christian Freese1,*, Alistair N Garratt2, Falk Fahrenholz1and Kristina Endres1
1 Institute of Biochemistry, Johannes Gutenberg-University, Mainz, Germany
2 Department of Neurosciences, Max-Delbru¨ck-Centre, Berlin, Germany
Neuregulin-1 (NRG-1) belongs to a family of growth
factors that transduce cellular signals by binding to
ErbB receptors [1,2] At least sixteen different gene
products of NRG-1 have been identified [3,4], which
display a wide range of functions in the developing as
well as in the adult organism Besides organs such as
the heart [5,6] or breasts [7], certain isoforms of
NRG-1 mediate important properties in the central
and peripheral nervous system: synapse formation [8]
and transmission [9], expression of neurotransmitter
receptors [10–12] and synaptic plasticity [13]
Addi-tionally, general features of neurones or Schwann
cells, such as proliferation, differentiation, migratory processes and regeneration, depend on NRG-1 activity [14–17]
Although some of these functions are restricted to the developing embryonic brain, expression of NRG-1
or at least some of its isoforms [18,19] and the ErbB receptors [13,17,20,21] persists throughout the adult rodent and human nervous system Within hippo-campal synapses of adult mice, for example, NRG-1b
is implicated in activity dependent remodulation by reversing long-term potentiation [22] Moreover, it induces neurite extension and arborization of primary
Keywords
Alzheimer; ErbB; metalloproteinase;
myelination; shedding
Correspondence
K Endres, Institute of Biochemistry,
Johannes Gutenberg-University,
Johann-Joachim-Becherweg 30,
55128 Mainz, Germany
Fax: +49 6131 3925348
Tel: +49 6131 3926182
E-mail: endres@uni-mainz.de
*Present address
Institute of Pathology, Johannes
Gutenberg-University, Mainz, Germany
(Received 28 August 2008, revised 22
December 2008, accepted 6 January 2009)
doi:10.1111/j.1742-4658.2009.06889.x
Although ADAM10 is a major a-secretase involved in non-amyloidogenic processing of the amyloid precursor protein, several additional substrates have been identified, most of them in vitro Thus, therapeutical approaches for the prevention of Alzheimer’s disease by upregulation of this metalloproteinase may have severe side effects In the present study,
we examined whether the ErbB receptor ligand neuregulin-1, which is essential for myelination and other important neuronal functions, is cleaved by ADAM10 Studies with b- and c-secretase inhibitors, as well
as with the metalloproteinase inhibitor GM6001, revealed an inhibition of neuregulin-1 processing in human astroglioma cell line U373; however, specific RNA interference-induced knockdown of ADAM10 remained without effect In vivo investigations of mice overexpressing either ADAM10 or dominant negative ADAM10 showed unaltered cleavage of neuregulin-1 compared to wild-type animals As a consequence, the mye-lin sheath thickness of peripheral nerves was unaffected in mice with altered ADAM10 activity Thus, although the b-secretase BACE-1 acts as
a neuregulin-1 sheddase, ADAM10 does not lead to altered neuregulin-1 processing either in cell culture or in vivo Adverse reactions of an ADAM10-based therapy of Alzheimer’s disease due to neuregulin-1 cleav-age are therefore unlikely
Abbreviations
APLP, amyloid precursor-like protein; APP, amyloid precursor protein; APPs, soluble APP fragment; DAPT,
N-[N-(3,5-difluorophenacetyl)-L -alanyl]-S-phenylglycine t-butyl ester; NRG-1, neuregulin-1; RNAi, RNA interference.
Trang 2cultures derived from adult murine hippocampi [21].
There is substantial genetic evidence that single
nucleo-tide polymorphisms of NRG-1 are associated with the
pathogenesis of schizophrenia [23–25] In addition,
NRG-1 has been found to be involved in the
patho-genesis of other diseases such as multiple sclerosis
[26,27] or breast cancer [7]
How proteins derived from the gene for NRG-1
ful-fil their different functions exactly remains elusive:
iso-forms of type I and III exist as transmembrane iso-forms
or can be proteolytically processed [8,28–30] to release
soluble fragments It is not known in detail whether
the transmembrane protein or its proteolytic products
are mainly responsible for the different functions
Because recombinant soluble NRG-1 often is sufficient
to induce morphological or biochemical phenotypes
[21,31] and shedding of NRG-1 is activity dependent,
as shown for electrically stimulated neurones [8], an
important role of the cleavage fragments is implicated
The proteinases involved in NRG-1 proteolysis have
been partly characterized: cleavage by the amyloid
precursor protein (APP)-processing c-secretase [32,33]
and, more recently, b-secretase BACE-1 [29,34] was
analyzed both in vitro as well as in vivo Additional
data have also been reported with respect to
metallo-proteinase-derived proteolysis of NRG-1 isoforms
For example, ADAM19 was shown to participate in
NRG-1-b shedding, whereas NRG-1-a2 was not
affected by coexpression of this enzyme [35] Cleavage
of the a2 isoform of NRG-1, on the other hand, was
impaired in fibroblasts with catalytically inactive
ADAM17 [30]
b- and c-secretase are responsible for processing of
the Alzheimer associated APP and its paralogues
amy-loid precursor-like protein (APLP) 1 and APLP2 [36]
Furthermore, the distribution of NRG-1 and the
local-ization of its receptor ErbB4 have been found to be
altered in Alzheimer’s disease patients [19,37] and a
mouse model of the disease [19] ADAM10 was found
to act as a-secretase in vitro and in cultured cells
[38,39] It competes with BACE-1 for the substrate
APP and is able to prevent the formation of Ab
pla-ques in a mouse model of the disease [40] Moreover,
ADAM10 restores long-term potentiation and
increases cognitive function in transgenic mice [40,41]
and enhances cortical synaptogenesis [42] Due to the
overlap of substrate specificity of BACE-1 and
ADAM10 with respect to substrates such as APP or
the APLPs and partial phenotypic overlap of
ADAM10 and NRG-1 knockout mice [43–45], it was
considered important to investigate the possible role of
ADAM10 in NRG-1 processing in cells and in the
living animal
Results
Identification of NRG-1 isoforms expressed in the human astroglioma cell line U373
The expression and processing of NRG-1 was described previously in different astroglioma cell lines [31] Therefore, we chose the human astroglioma cell line U373 to examine the relevance of ADAM10 for NRG-1 shedding The investigated cell line stably overexpresses the human neuron specific APP isoform
695 to provide an appropriate control substrate for a- as well as b- and c-secretases [39,46]
Recently, sixteen different isoforms of NRG-1 gen-erated by alternative promoter usage, transcription initiation sites or splicing [4,47,48] have been described, and a wide variety of these isoforms are found in brain-derived cell types [49–51] To charac-terize the isoforms present in the U373 cell line, we performed RT-PCR with domain specific primers [52,53] Type I as well as type III specific PCR prod-ucts (schematically shown in Fig 1A: immunoglobu-lin-like domain and glycosylation site or cysteine rich domain sequences) were produced, whereas those characteristic of the type II Kringle domain coding region were not detectable (Fig 1B) Both a- as well
as b-type indicating PCR products were generated
We amplified the juxtamembrane region coding sequence with primers independent of a- or b-type (primers jD_for and TM_rev; Table 1) and subcloned the resulting DNA fragments into pUC19 Sequencing analysis revealed that seven out of eight clones were the a2-type, whereas only one was identified as b2 (for sequences, see Fig 1B; NM_013964 and NM_13957) Hence, the predominant NRG-1 isoform present in U373 cells is the a2-type with respect to the juxtamembrane region Because ADAMs such as ADAM10 cleave their substrates in close proximity to the membrane, knowledge of this region of the puta-tive substrate NRG-1 in the investigated cell line was mandatory
On the protein level, the NRG-1 antibody against the C-terminal domain detected a prominent band of approximately 90 kDA in the lysate of U373 cells (Fig 1C) This is consistent with full length NRG-1 in its glycosylated state [31] for a panel of glioma cells Protein bands with a higher molecular weight might indicate the immature proform and the band with a reduced molecular weight might represent an incom-pletely glycosylated intermediate Additionally, between 40 and 50 kDa, two C-terminal fragments were detectable After serum free incubation of cells for 4 h, a band of approximately 60 kDa (N-terminal
Trang 3fragment) was detected in cell supernatants using the
pan-NRG-1 antibody against the ectodomain, which
recognizes a- as well as b-isoforms (Fig 1C) This
pro-tein reveals a slightly lower molecular weight
com-pared to the results obtained by Ritch et al [31] where
secreted NRG-1 had a molecular mass of 70 kDa
Because at least two Asn residues and 11 Thr⁄ Ser
resi-dues were identified as potential sites for N- or
O-gly-cosylation of NRG-1 [54], the deviation in the size of
the soluble protein fragment may depend on different glycosylation patterns in the investigated cell lines In mouse brain membranes, a panel of proteins in the approximate range of 160–70 kDa was observed (Fig 1C), which corresponds to NRG-1 species described for mouse brain as well as human brain material [19] Similar to cell supernatants, the soluble fraction of mouse brain contained a secreted form of NRG-1 of 55–60 kDa
Fig 1 Isoforms of NRG-1 expressed in the human astroglioma cell line U373 and mouse brain (A) In general, three major types of NRG-1 are generated, which all share an EGF-like domain; further variation is achieved through differences in the sequences of the C-terminal part of the EGF domain (a or b) isoforms, and the juxtamembrane region (e.g a2 or b1 isoforms) and other domains such
as the type II specific Kringle or the type III specific cysteine-rich domain (B) To identify mRNA species of NRG-1 present in the human astroglioma cell line U373, RT-PCR was performed A sample lacking RNA was used as a no template control (NT) and a GAPDH sequence was amplified for the reaction control (C) NRG-1 protein expression in U373 cells and mouse brain was analyzed using the antibody against the C-terminus or against the extracellular domain Cell lysate or the membrane fraction from mouse brain was subjected to 4–12% NuPAGE and cell supernatants (medium) or soluble proteins from mouse brain were subjected to 8% SDS ⁄ PAGE NRG-1 protein species (FL, full length; NTF, N-terminal fragment; CTF, C-terminal fragment) were visualized after transfer onto poly(vinylidene difluoride) membrane.
Trang 4Table 1 Primer sequences used for NRG-1 isoform analysis.
Length of amplificate (bp)
NRG-TM_rev
TGAAAGACCTTTCAAACCCCTC GTTTTGCAGTAGGCCACCAC
Approximately 200 (depending on isoform) Immunoglobulin domain
(type I and II)
NRG-IG_for NRG-TM_rev
GCCAGGGAAGTCAGAACTTC GTTTTGCAGTAGGCCACCAC
543
Glycosylation sites (type I) NRG-Glyc_for
NRG-TM_rev
CCACAGAAGGAGCAAATACTTC GTTTTGCAGTAGGCCACCAC
339
NRG-Kringle_rev
AGGAGGAGGAGTGGTGCTG GTCCCCAGCAGCAGCAGTA
239
Cysteine rich domain (type III) NRG-CRD_for
NRG-CRD_rev
GAGGTGAGCCGATGGAGATTTA CCTCTCAGGCGCTCAGCTTC
219
NRG-a_rev
TCTCCGGCGAGATGTCCGA GCTCCAGTGAATCCAGGTTG
668
NRG-Beta_rev
TCTCCGGCGAGATGTCCGA GGCAGCGATCACCAGTAAAC
677
GAPDH_rev
GAAGGGCTCATGACCACAGTCCAT TCATTGTCGTACCAGGAAATGAGCTT
450
Fig 2 Proteolytical processing of APP and NRG-1 in U373 cells U373 cells overexpressing human APP695 were incubated with inhibitors for b-secretase, c-secretase or metalloproteinases, or stimulated with phorbol 12-myristate 13-acetate Proteolytic processing products of APP or NRG-1 were detected in culture supernatants after precipitation or in lysed cells with appropriate antibodies (A) Cells were incubated with the tripeptidic inhibitor of the b-secretase (25 l M ) and shedded APPs-b or NRG-1 was visualized by western blotting (B) Full length pro-tein (FL) or C-terminal membrane tethered fragments (CTF) of either APP or NRG-1 were detected in cell lysates after an incubation period
of 48 h with 2 l M DAPT (C) Phorbol 12-myristate 13-acetate (PMA) (1 l M , 4 h) or GM6001 (10 l M , 26 h) were added to the cells to investi-gate the influence of metalloproteinases on secretion of NRG-1 ectodomain (NTF, N-terminal fragment) in U373 cells APPs-a served as a control All blots show samples from solvent-treated cells in lanes 1 and 3, whereas lanes 2 and 4 show samples from compound-treated cells Blots are representative for at least three independently performed experiments per treatment Quantifications display the mean ± SD; values from solvent-treated cells were set to 100% (Student’s t-test: ***P < 0.001; **P < 0.01; *P < 0.05).
Trang 5Proteolytical processing of NRG-1 in the human
astroglioma cell line U373
The tripeptidic b-secretase inhibitor II led to an 80%
reduction of soluble b-secretase cleaved APP (APPs-b)
in cell culture supernatants and also diminished
signifi-cantly the 60 kDa soluble NRG-1 in cell conditioned
medium (N-terminal fragment; Fig 2A) Furthermore,
the c-secretase inhibitor
N-[N-(3,5-difluorophenacetyl)-l-alanyl ]-S-phenylglycine t-butyl ester (DAPT), which
induced accumulation of C-terminal fragments of APP
in cell lysates (C-terminal fragment; Fig 2B), increased
the NRG-1 C-terminal fragment of approximately
50 kDa six-fold as compared to solvent-treated cells
These results demonstrate cleavage of NRG-1 in U373
cell line by both b- and c-secretase
Phorbol 12-myristate 13-acetate, a known inducer of
shedding events, significantly elevated soluble APPs-a,
as well as the soluble N-terminal fragment of NRG-1
in those cells, by 200% and 150% (Fig 2C) For this
reason, we analyzed metalloproteinase dependent
shed-ding of NRG-1: APPs-a that acted as a control was
reduced to 40% by the broad spectrum
metalloprotein-ase inhibitor GM6001 as compared to solvent-treated cells, and NRG-1 cleavage also was reduced signifi-cantly, although to a lower extent (65% of control cells; Fig 2C) Therefore, metalloproteinases appear to
be involved in NRG-1 processing in the astrocytoma cell line U373, as previously described for other cell lines [28,55]
RNA interference (RNAi)-induced knockdown of ADAM10 has no influence on NRG-1 shedding Because GM6001, which was used for inhibitory stud-ies, is a broad spectrum inhibitor of MMPs as well as ADAMs, we chose the RNAi approach to analyze in particular the role of ADAM10 in NRG-1 cleavage As
a control for unspecific RNAi-induced effects, MMP2 knockdown was examined as well RNAi treatment tar-geted against endogenous ADAM10 of the U373 cells resulted in a 60% reduction of mature ADAM10, whereas MMP2 targeted oligomers had no influence (Fig 3A) The decrease of ADAM10 due to RNAi was accompanied by a 30% decrease in APPs-a shedding (Fig 3B) serving as an internal control Because APP is
Immature Mature
Fig 3 Influence of siRNA mediated knock-down of ADAM10 on APP or NRG-1 processing in U373 cells U373 cells were transfected with a set of RNA oligomers targeted to ADAM10 (AD)
Mock-transfect-ed cells (C) or cells transfectMock-transfect-ed with RNA oligomers against MMP2 (M) were used as controls Forty-eight hours after transfection, cells were investigated with respect to ADAM10 and products of APP or NRG-1 proteolysis (A) The mature and immature forms of ADAM10 in the cell lysates were detected by western blotting and the mature, catalytically active form of the enzyme was quantified (B) APPs-a was enriched by trichloroacetic acid precipitation and visualized by the specific antibody 6E10 (C) Secreted (NTF, N-terminal frag-ment) and membrane bound NRG-1 species (CTF, C-terminal fragment) were detected in cell supernatants or lysates All western blottings show two sets of independent samples Quantifications are based on four independent experiments and show the mean ± SD; values from mock-transfected cells were set to 100% (one-way analysis of variance ⁄ Bonferroni post hoc test:
***P < 0.001; **P < 0.01).
Trang 6not only a substrate for ADAM10, but also, for
exam-ple, TACE, the absolute effect of ADAM10 knockdown
was small but reached significance By contrast, for
NRG-1, we observed no alteration of soluble NRG-1 in
cell culture supernatants, as well as for the
membrane-bound protein species Because of a potential
compensa-tion of reduced NRG-1 cleavage by other secretases, we
cannot exclude the possibility that ADAM10 might
have an effect on proteolytic processing of NRG-1 in
U373 cells but, if this is the case, ADAM10 at least is
not a major sheddase of this protein (Fig 3C)
In vivo effect of ADAM10 on NRG-1 proteolysis
Because ADAM10 was not implicated in the shedding
of distinct NRG-1 isoforms of cultured human
astro-glioma cells (a2 and b2; Fig 1B), we analyzed NRG-1
processing in ADAM10 overexpressing mice to take
into account all of the expressed isoforms Two
trans-genic mouse lines with different expression levels of
ADAM10 (moderate, ADAM10mo; high, ADAM10hi)
and a mouse line transgenic for a dominant negative
ADAM10 mutant (ADAM10dn) were included in this
investigation All mouse lines have been examined in
detail elsewhere with respect to APP processing,
learn-ing and behaviour [40,41,56] The expression of the proteinase itself is illustrated in Fig 4A (lower part)
In soluble protein fractions of brains derived from the three transgenic lines, the amount of the N-terminal fragment of NRG-1 (approximately 60 kDa; Fig 4) was not changed compared to the wild-type Addition-ally, neither full length NRG-1, nor C-terminal frag-ments in the brain membrane fraction were influenced
by an altered ADAM10 amount or activity (Fig 4)
We therefore conclude that, in vivo, the proteolytic processing of NRG-1 does not depend on the a-secre-tase ADAM10
For further confirmation of these findings, we exam-ined the myelination of peripheral nerves in ADAM10 transgenic mice and mice overexpressing dominant negative ADAM10 Because myelination strongly depends on NRG-1-ErbB signalling of Schwann cells and neurones [57], any relevant change of this pathway induced by altered ADAM10 activity should be obser-vable as a physiological consequence Again, the ADAM10 transgenes remained without effect in all investigated mouse lines (Fig 5A) G-ratios of ADAM10mo as well as of ADAM10dn mice at postnatal day 17 were identical to nontransgenic litter-mates Furthermore, Akt-phosphorylation, which also
Fig 4 Processing products of NRG-1 in ADAM10 transgenic mice Soluble and membrane tethered fractions of NRG-1 from brains of ADAM10mo, ADAM10hi and ADAM10dn mice were detected by western blotting with antibodies against the N- (NT) or the C-terminus (CT)
of the protein Nontransgenic littermates (Wt, wild-type) were used as controls Each western blot for NRG-1 shows samples from two indi-viduals: lanes 1 and 3 are from wild-type animals and lanes 2 and 4 are from transgenic mice For ADAM10 (detection by HA-antibody), one exemplary blot from the brain membrane fraction of four individuals is shown The proform of the proteinase is indicated by a black arrow head and the catalytically active form is indicated by a grey arrow head (B) Protein bands with respect to shedded (N-terminal fragment; 60 kDa) or one exemplary C-terminal fragment (50 kDa) of NRG-1 were quantified and values from wild-type mice were set to 100% (mean ± SEM; n = 6 for each mouse line, P > 0.05).
Trang 7Fig 5 ADAM10 transgenic mice display no disturbance in peripheral myelination (A) Sciatic nerves of ADAM10mo, ADAM10dn and wild-type (Wt) mice (postnatal day 17) were analyzed for myelin sheath thickness by electron microscopy Two exemplary microscopic images are shown for each mouse line G-ratios were evaluated taking into account at least 350 individual axons per group (n = 3 animals for each group) (B) Myelination was analyzed in adult, aged ADAM10hi mice (15–17 months) in analogy to (A) Two electron microscopy images at two different magnifications (see scale bars) of transgenic mice and age-matched control mice are shown Tomacula-like structures are indicated by black arrows.
Trang 8is partly controlled by NRG-1 signaling [31,58,59], was
unaffected in both mouse lines (Fig 6) In adult mice
with a high expression level of ADAM10
(ADAM10hi), G-ratios were also unaltered (Fig 5B),
but tomacula-like structures (local myelin thickenings
[60]) were observed Additionally, in the mouse line
with higher ADAM10 expression (ADAM10hi),
Akt-phosphorylation was significantly reduced to 40%
compared to wild-type mice This probably reflects
effects that do not depend on NRG-1 cleavage
Discussion
The data obtained in the present study for the human
astroglioma cell line U373 clearly reveal BACE-1 and
c-secretase dependent shedding of the endogenous
ErbB receptor ligand, which we identified
predomi-nantly as type a2-NRG-1 and, to a lesser extent, as the b2 isoform Additionally, GM6001, a broad spec-trum metalloproteinase inhibitor, was able to reduce NRG-1 shedding but a specific knockdown of ADAM10 by RNAi remained without any effect within the cellular system Therefore, the present study demonstrates that ADAM10 is not a major sheddase
of neuregulin-1 and enhancement of ADAM10 will probably have no side effects due to NRG-1 cleavage Because catalytically active ADAM10 is found on the plasma membrane [38] and neuregulin-1b1 cleav-age, for example, is restricted to the Golgi apparatus [28], it is plausible that distinct localization of ADAM10 and NRG-1 might inhibit a functional sub-strate–proteinase interaction Furthermore, NRG-1 is mainly found in cholesterol rich lipid rafts [61,62], favouring its role as a BACE-1 substrate, whereas ADAM10 and its catalytic activity (at least for APP) were shown to be localized in cholesterol-poor nonraft regions of the membrane [39] Nevertheless, a possible
in vivo relevance of ADAM10 to NRG-1 shedding required investigation due to the fact that NRG-1 pro-teolysis also depends on, for example, electric stimula-tion of cells [8], which might be accompanied by translocation within the cell Furthermore, the animal model offers a more complex representation because of the wide variety of cells that express NRG-1 and which might interact
Reconstitution experiments with transfection of ADAM10 in ADAM17) ⁄ ) embryonic mouse fibro-blasts [55] suggested only a minor influence of ADAM10 on neuregulin-1 shedding, but any positive proof in the living animal was still missing Therefore,
we investigated neuregulin-1 processing in mice with postnatal expression of ADAM10 or its dominant neg-ative variant The Thy.1-promoter driven expression of both ADAM10-constructs [40] occurs at postnatal day 1 (data not shown) Thy.1-based expression in general is predominantly found in postmitotic neuro-nes of the perinatal period, but also occurs in dorsal root ganglia and in spinal cord [63] Hence, the animal model is sufficient to study early ontogenetic phenom-ena after birth without disturbances due to impeded embryonic development in the central or peripheral nervous systems
In a recent study, the age-dependency of NRG-1 cleavage by BACE-1 was demonstrated [64] Although the accumulation of full length neuregulin-1 in BACE) ⁄ ) mice aged 15 days confirmed previous data [29,34], mice at postnatal day 30 or even older (2 years) showed no abnormalities with respect to neu-regulin-1 processing In the case of ADAM10, investi-gations of adult mice moderately overexpressing
Fig 6 Akt-phosphorylation in ADAM10 transgenic mice (A)
Total-Akt and phospho-Total-Akt were detected by western blotting in soluble
fractions of brains from ADAM10mo, ADAM10hi and ADAM10dn
mice Nontransgenic littermates (Wt, wild-type) were used as
con-trols (B) Phospho-Akt was normalized by total-Akt and quotients
from wild-type mice were set to 100% Values represent
the mean ± SEM (n = 4 for each mouse line; one-way analysis of
variance ⁄ Bonferroni post hoc test: **P < 0.01).
Trang 9ADAM10 or its dominant negative variant resulted in
totally unchanged amounts of NRG-1 processing
products
Therefore, the influence of ADAM10 on NRG-1 was
additionally analyzed in young mice (postnatal day 17)
by the status of peripheral nerve ensheathment In the
second postnatal week, myelination is almost finished in
mice (central nervous system [65]; peripheral nervous
system [66]); therefore, alterations should be apparent
However, neither moderate ADAM10 overexpressing
mice, nor mice with a restriction of enzyme activity by
dominant negative ADAM10, revealed differences in
axon myelination parameters compared to wild-type
littermates at postnatal day 17
Surprisingly, adult mice with high levels of
ADAM10 overexpression showed myelin infoldings
(tomacula-like structures) This observation has not
been made in the context of reduced or enhanced
NRG-1 signalling in mice [67] We therefore suggest
that mechanisms beside NRG-1 signal transduction
might be responsible for the neuropathological
pheno-type It will be interesting to analyze these
observa-tions in future studies
Additionally, Akt phosphorylation, a consequence
of NRG-1-ErbB signalling [31,59], was unaltered by
moderate overexpression or by inhibiting ADAM10
activity by its negative mutant form However, mice
with high overexpression of ADAM10 showed a strong
decrease of phosphorylated Akt compared to
non-transgenic mice This observation may relate to recent
findings demonstrating that a high level of ADAM10
overexpression in the mouse increases susceptibility to
kainate-induced seizures and neuronal damage [56],
whereas the neuroprotective properties of ADAM10
were only evident in mice with APP overexpression
In conclusion, ADAM10 was excluded both in cell
culture and in the animal model as a major candidate
secretase for neuregulin-1 shedding We cannot rule
out that other secretases, such as TACE or ADAM19,
which were identified formerly as NRG-1 sheddases
[28,30,55], compensate for the lack of ADAM10 in
RNAi-treated cells or animals with overexpression of
the dominant negative mutant In breast cancer cells,
ADAM10 was described to mediate the shedding of
the receptor ErbB2 [68]; therefore, an influence on
NRG-1-ErbB signalling could in principal have also
occurred by ErbB2 cleavage in our transgenic mice
However, because we did not observe an influence on
myelination in ADAM10 transgenic mice, this
observa-tion might be restricted to tumour cells
We also cannot exclude that, in non-neuronal
tis-sue, embryonic development or pathological stages
ADAM10 itself, or cleavage products of its other
sub-strates, might be involved in NRG-1-ErbB cross-talk
In summary, however, we present evidence demonstrat-ing that, in the healthy early postnatal and adult mouse, moderate alterations in the amount of ADAM10 do not interfere with neuregulin-1 signalling Accordingly, ADAM10 will have no impact on down-stream physiological functions such as nerve remyelina-tion or the schizophrenia-resembling psychiatric changes as observed for BACE-1 knockout mice [34,69] The results obtained in the present study there-fore suggest that a moderate upregulation of ADAM10 expression and its a-secretase activity with a preventive
or therapeutical intention is not impaired by side effects resulting from the NRG-1-ErbB signalling network
Experimental procedures
Antibodies, inhibitors and RNAi oligomers
The primary antibodies used were: 6E10 for the detection
of APPs-a (Senetek, St Louis, MO, USA; dilution
1 : 1000), anti-neuregulin-1 (H-210; dilution 1 : 200) for the detection of secreted NRG-1 fragments, anti-neuregulin-1a⁄ b1 ⁄ 2 (C-20; dilution 1 : 500) (both Santa Cruz Biotech-nology, Santa Cruz, CA, USA) for the detection of membrane bound NRG-1, anti-ADAM10 (Chemicon, Temecula, CA, USA; dilution 1 : 1000) for the detection of the proteinase in cells and 6687 (C Haass, LMU Munich, Germany) for the detection of full length APP and C-termi-nal protein fragments Anti-P-Akt and anti-total Akt were purchased from Cell Signaling [PhosphoPlus Akt (Ser473) Antibody Kit; Cell Signaling, Danvers, MA, USA] Overex-pressed ADAM10 in mouse brain membranes was visual-ized by HA-antibody Y-11 (Santa-Cruz Biotechnology) The secondary antibodies were coupled to alkaline phos-phatase (Tropix, Bedford, MA, USA; dilution 1 : 10000) or horseradish peroxidase (Pierce, Rockford, IL, USA; dilution 1 : 3000) and were used in combination with their substrates CDP-Star (Tropix) or SuperSignalECL (Pierce) The b-secretase-inhibitor II (Calbiochem, Bad Soden, Germany) was applied at a concentration of 25 lm and the c-secretase inhibitor DAPT (B Schmitt, Clemens Scho¨pf-Institute of Organic Chemistry and Biochemistry, Tech-nische Universita¨t Darmstadt, Germany) was applied at a concentration of 2 lm GM6001 (Calbiochem, San Diego,
CA, USA) was used at a final concentration of 10 lm and phorbol 12-myristate 13-acetate (Sigma, Deisenhofen, Ger-many) was used at a concentration of 1 lm All substances were dissolved in dimethylsulfoxide as stock solutions For the RNAi experiments, the Stealth RNAis ADAM10 HSS165, HSS166, HSS167 and MMP2 HSS106612, HSS106613, HSS106614 (Invitrogen, Karlsruhe, Germany) were used Transfections were performed with Opti-MEM and Lipofectamine2000 (Invitrogen)
Trang 10RNA preparation and RT-PCR
The RNA of U373 cells was isolated by using confluent 6 cm
culture plates and the RNA isolation kit with on-column
DNA digestion as recommended in the manufacturer’s
pro-tocol (Macherey-Nagel, Du¨ren, Germany) Four hundred
nanograms of RNA were reverse transcribed in a 20 lL
reac-tion volume by the reverse-it-RT-PCR-kit from ABgene
(Hamburg, Germany) with intron-spanning specific primers
(0.2 lm each) Amplificates were analyzed on 1% agarose
gels and GAPDH amplification served as a control for the
RT-PCR reaction and PCR conditions Primer sequences
and the amplificate length are provided in Table 1
The amplificates from RT-PCR with primers
NRG-jD_for and NRG-TM_rev were ligated into pUC19
(Fer-mentas, St Leon-Rot, Germany) and plasmid DNA from
eight positive clones (identified by blue–white selection) was
sequence analyzed with M13 universal primer
Preparation of mouse brain samples
The generation of transgenic mice has been described
previously [40] Seven- to 10-week-old mice were sacrificed,
the brains were dissected and stored on dry ice Ice-cold
Tris buffer (20 mm Tris⁄ HCl, pH 8.5) containing proteinase
inhibitors (Inhibitor Complete Mini; Roche Diagnostics
Corp., Mannheim, Germany) was added and tissue was
homogenized in a tissue lyser (Eppendorf, Hamburg,
Ger-many) with a frequency of 20 Hz for 2 min The
superna-tants resulting from centrifugation at 13 500 g for 1.75 h
were used for the detection and quantification of soluble
NRG-1 NRG-1 membrane bound full length protein and
membrane bound NRG-1 fragments, as well as the
protein-ase ADAM10 itself, were detected in the membrane
frac-tions prepared from centrifugation pellets
For total-Akt and phospho-Akt detection, brain
hemi-spheres were homogenized in lysis buffer supplemented with
additional phosphatase inhibitors (2.5 mm
Na-pyrophos-phate, 1 mm b-glycerophosphate and 1 mm Na3VO4) and
soluble proteins were isolated from membrane fractions by
centrifugation at 20 800 g for 20 min
Cell culture, treatment with inhibitors and RNAi
silencing
U373 cells overexpressing human wild-type APP were
main-tained in MEM (Sigma, Taufkirchen, Germany)
supple-mented with 10% fetal bovine serum, 1% sodium pyruvate
and 1% glutamine
To inhibit b-secretase or metalloproteinases, cells were
pretreated with the appropriate inhibitor
(b-secretase-inhibi-tor II or GM6001) for 22 h Then, the serum containing
culture medium was removed and medium without serum
supplemented with fatty acid free BSA (1 mgÆmL)1) and
fresh inhibitor was added for an additional 4 h Phorbol 12-myristate 13-acetate-induced shedding was performed for 4 h in serum free BSA supplemented medium For inhi-bition of c-secretase, cells were treated with DAPT for 48 h
in culture medium
For RNAi experiments, cells were transfected with
750 pmol of RNAi oligomers (250 pmol each) in six-well plates After 5 h of transfection, the medium was replaced
by culture medium After 44 h, cells were covered with serum-free medium and incubated for 4 h for collection of secreted proteins and cell lysates
Western blotting
Proteins of cell culture supernatants were precipitated with trichloroacetic acid and normalized by the protein content
of the cell lysates Adequate amounts of soluble or mem-brane tethered proteins were separated on 8% SDS-gels or 4–12% Bis–Tris NuPAGE gels (Invitrogen) and blotted on
a poly(vinylidene difluoride) membrane or nitrocellulose (total-Akt and phospho-Akt) Proteins were then detected with the appropriate primary antibodies Chemiluminescent signals from alkaline phosphatase or horseradish peroxidase coupled secondary antibodies were visualized with a charge-coupled device camera and the software versa doc (Bio-Rad, Munich, Germany) and were quantified with aida3.5 (Raytest, Straubenhardt, Germany)
Electron microscopy and G-ratio determination
Seventeen-day-old or adult transgenic mice (15–17 months old) and nontransgenic littermates were perfused with NaCl⁄ Picontaining 60 lgÆmL)1 heparin followed by 2.5% glutaraldehyde⁄ 4% parafomaldehyde in 0.1 m phosphate buffer Afterwards, sciatic nerves were removed, postfixed and contrasted with osmium tetroxide and processed for electron microscopy [70] biovision software (Soft Imaging System GmbH, Mu¨nster, Germany) was used for determi-nation of the G-ratio by measuring the inner and outer myelinated fibres
All animal procedures were performed according to the German guidelines for the care and the use of laboratory animals and in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC)
Acknowledgements
We thank C Griffel (MDC, Berlin) for excellent tech-nical support in the analysis of sciatic nerve myelina-tion; A Schro¨der (ZVTE, Mainz) for coordination of animal husbandry and M Willem (LMU, Munich) for fruitful discussion This work was supported by grants from the DFG priority program 1040 (to F.F.)