BACKGROUND
accumulation of amyloid β-peptides  in the brain is believed to contribute to the development of alzheimer disease .
soluble oligomeric forms of aβ are neurotoxic  <cit> .
aβ, a 40– <dig> amino acid-comprising proteolytical fragment of the amyloid precursor protein , is released from app by sequential cleavages via β- and γ-secretases.
however, the predominant route of app processing consists of successive cleavages by α- and γ-secretases.
alpha-secretase attacks app inside the aβ sequence, and therefore prevents formation of neurotoxic aβ.
in addition, the soluble n-terminal domain of app  is released, which has neurotrophic and neuroprotective properties  <cit> , and enhances ltp  <cit> .
in behavioral paradigms, appsα was demonstrated to improve memory in normal and amnesic mice  <cit> .
reduced amounts of appsα were detected in the cerebrospinal fluid of ad patients  <cit> .
proteinases of the adam  family are main candidates for physiologically relevant α-secretases, and we demonstrated that adam <dig> has α-secretase activity in vitro and in cultured cells  <cit> .
adam10-deficient mice have been generated  <cit> , but their early lethality at day e <dig>  prevents a reliable analysis of adam10's α-secretase function in vivo, especially in neuronal cells.
to investigate whether an increase in activity of putative α-secretases in vivo prevents plaque formation and cognitive deficits, we generated transgenic mice overexpressing either the α-secretase adam <dig>  or the catalytically inactive adam <dig> mutant   <cit> .
neuronal overexpression of adam <dig> had no detrimental effects on adam <dig> single-transgenic mice: these animals exhibited normal behavioral abilities  <cit> .
we found that a moderate neuronal overexpression of adam <dig> in mice carrying the human app mutation  increased the secretion of appsα, reduced the formation of aβ peptides, and prevented their deposition in plaques.
functionally, impaired long-term potentiation and cognitive deficits were alleviated.
expression of dominant-negative adam <dig>  in app mice  led to reduction of appsα and to enhancement of the number and size of amyloid plaques in the brain  <cit> .
histological analyses of mono-transgenic adam <dig> mice revealed an increase in cortical cholinergic, glutamatergic and gabaergic presynaptic bouton densities in  <dig> months old mice; the cholinergic presynaptic bouton density remained elevated even during aging in adam <dig> mice  <cit> .
in addition to their metalloproteinase domain, adams contain a disintegrin domain as a modulator of cell-cell and cell-matrix interactions  <cit> .
as adam <dig> itself has been reported to be a substrate for ectodomain shedding by adam <dig> and subsequent cleavage by γ-secretase, the c-terminus of adam <dig> may represent a notch-like signaling molecule  <cit> .
thus, independent of the catalytic activity of adam <dig>  which has been implicated in notch and β-catenin signaling, adam <dig> may also modulate gene expression via other domains.
to analyze the influence of adam <dig> and its dominant-negative form  on the gene expression profile of the central nervous system , we investigated adam <dig> and dnadam <dig> mice.
we included in our study the double-transgenics adam10/app and dnadam10/app.
since app mice show early phenotype changes , we investigated the gene expression in  <dig> months old mice.
methods
animals
animal husbandry was performed in accord with the guidelines of the german council on animal care.
all mouse strains  analyzed in this study have been described previously  <cit> .
the expression level of transgenic mature adam <dig> is 30% above endogenous levels and in dnadam <dig> mice the expression of the catalytically inactive adam <dig> mutant is sevenfold above endogenous adam <dig>  <cit> .
adam <dig> activity was determined in previous studies  <cit>  by quantitation of the app cleavage product appsα.
in adam <dig> overexpressing mice the catalytic activity of adam <dig> against its substrate app was increased to about 250%.
in mice overexpressing dnadam <dig>  the endogenous app cleavage activity was reduced to about 25% as compared to app mice  <cit> .
for the first experimental series of the present study, female adam <dig>  dnadam <dig> and fvb/n wild-type mice were investigated; for the second series, female and male adam10/app, dnadam10/app and app mice were compared.
in each case, brains of three  <dig> months old animals of each group were dissected and stored in rna-later  at -80°c to prevent rna degradation.
rna preparation and microarray analyses
total rna from whole mouse brains was isolated by using the rneasy kit , including on-column dnase i digestion according to the manufacturer's recommendations.
the quality of isolated rna was controlled by the lab-on-chip-system bioanalyser  <dig> .
the expression-profiling analysis for mono-transgenic mice  was carried out at rzpd .
samples from double-transgenic mice  were analyzed at the microarray facility .
in all cases, the mouse genome  <dig>  <dig>  array  containing  <dig> probe sets of  <dig> genes was used for mrna expression profiling.
statistical analysis and gene annotations
for the first series   <dig> gene chip arrays and for the second series   <dig> gene chip arrays were analyzed.
data mining was performed by using the chipinspector analysis software , which identifies significant changes based on single probes.
the corresponding transcripts were then assigned after a user-defined number of significant probes.
for all analyses, a transcript coverage greater than three probes was chosen.
by this strategy, annotation errors and errors caused by the existence of alternative transcripts are reduced.
after total intensity normalization of each array, significantly changed genes were determined by significance analysis of microarrays   <cit> , using the exhaustive comparison mode at a false discovery rate  of  <dig> % for double-transgenic, and  <dig> % for mono-transgenic mice.
for separate analysis of samples from double-transgenic female and male mice, a fdr of  <dig> % was chosen.
the resulting gene lists were restricted to the  <dig> most strongly regulated genes .
regulated genes were then analyzed with the bibliosphere software  and mapped to gene ontology  trees in order to identify their biological function.
for identification of over-represented go terms, the bibliosphere software calculates a z-score for each term.
the z-score represents the difference between observed and expected annotations, and is normalized to the standard deviation of a hypergeometric distribution.
only go terms with a z-score >  <dig> , which corresponds to a p-value of  <dig> , have been considered.
to identify transcripts which are affected by adam <dig> and dnadam <dig> overexpression in mono- and double-transgenic mice, we generated venn diagrams with sam-based gene lists.
the expression profile of selected significantly regulated genes from microarrays was represented by heat maps using the r statistical software .
hierarchical clustering was applied to investigate whether expression values can be separated according to experimental groups.
in this study, two heat maps were generated: one compared the expression profiles of genes in adam <dig> and dnadam <dig> mono-transgenic mice, as well as in fvb/n non-transgenic control mice; a second one compared the expression profiles of double-transgenics and app mice.
because the two series of expression arrays were measured in different laboratories, a global normalization procedure was needed to make them comparable.
the default background noise adjustment, provided by the affymetrix system, is based on the difference of perfect matching probes  minus mismatching probes .
due to unspecific binding, the global background adjustment method robust multi-array average  expression measure, which ignores the mm intensities, has been developed  <cit> .
because rma adjustment does not completely remove unspecific intensities  <cit> , an enhanced method denoted genechip rma  has been designed  <cit>  which considers the sequence of probes.
we performed background adjustment as well as quantile normalization for all data sets  with the gcrma method  by using the carmaweb interface  <cit> .
subsequently, an unpaired two-tailed student's t-test was applied for each respective gene to determine whether it is differentially expressed in the two sample groups.
since microarray analysis operates with large numbers of multiple comparisons, a false discovery rate-controlling method has to be applied.
therefore, by using the benjamini-hochberg  method, adjusted p-values were calculated  <cit> .
the gcrma method is also appropriate for detection of minor changes in gene expression, and was required for comparative analysis of mono- and double-transgenic mice, due to the low intensities of the microarrays from the first series  as compared to those of the second series .
by comparing data derived from mono- and double-transgenic mice, we analyzed global biological trends of adam <dig> and dnadam <dig> overexpression in fvb/n and fvb/n app strain backgrounds.
to identify transcripts which were commonly affected by appoverexpression in all double-transgenic mice, we generated a venn diagram with gcrma-based gene lists .
quantitative real-time rt-pcr
a two-step real-time reverse transcription -pcr was used to measure the expression of candidate genes.
isolated total rna  was used to synthesize cdna in a  <dig> μl reaction with the quantitect reverse transcription kit  according to the manufacturer's manual.
by adding water, the reaction volume was subsequently increased to  <dig> μl.
real-time rt-pcr was carried out in 96-well plates, using the  <dig> abi prism sequence detection system  and quantitect primer assays .
the primers for selected candidate genes are listed in table  <dig>
real-time rt-pcr reactions in a volume of  <dig> μl were performed in duplicate or triplicate under the following conditions:  <dig> μl of diluted cdna ,  <dig> μl 2× quantitect pcr master mix  and  <dig> nm of respective primer pair.
after the initial denaturing and enzyme activation step ,  <dig> cycles  were performed.
a single dna melting profile was observed in dissociation assay conditions demonstrating amplification of a unique product free of primer dimers.
for detection of hes <dig> in  <dig> day old mice a one step real-time rt-pcr was performed using the quantitect-sybr-green one-step-rt pcr-kit  and  <dig> ng rna in a reaction volume of  <dig> μl.
the specific primer pair was as follows: hes5rt_for 5'gaaaaaccgactgcggaagcc3' and hes5rt_rev 5'acggccatctccaggatgtc3'.
for data analysis, the threshold cycle  which indicates the relative abundance of a particular transcript, was calculated.
standard curves were generated by amplification of serially diluted cdna.
according to this method, the amount of all relevant genes was normalized to the amount of endogenous gapdh present in the same sample.
measured values from control samples  were set to 100%.
changes in gene expression are presented as the mean of alteration ± sd.
the data were analyzed for statistical significance using one-way anova .
western blotting
mouse brain tissue was stored on dry ice immediately after dissection.
ice-cold tris buffer  containing proteinase inhibitors  was added, and tissue was homogenized in a tissue lyser .
the supernatants resulting from centrifugation at  <dig> rpm for  <dig>  hours were separated on 14% sds-gels and transferred to nitrocellulose membrane by tank blot system .
for the detection and quantification of soluble fabp <dig> antibody ab <dig> , and the appropriate horseradish peroxidase-coupled secondary antibody  were used.
elisa
hemispheres of mouse brain were weighed, proteins extracted and calprotectin  was quantified as recommended by the elisa manufacturer .
in brief, tissue was homogenized in extraction buffer for  <dig> min at  <dig> hz in a tissue lyzer and extraction was performed for  <dig> min at 4°c under agitation.
after centrifugation  the supernatant and protein standards were added to microtiter plates in a total volume of  <dig> μl in duplicates.
incubation of the plate and measurement of optical densities at  <dig> nm were performed following the manufacturer's instructions.
the relative amount of calprotectin was calculated by division of background-corrected values by wet tissue weight.
RESULTS
microarray analysis of gene regulation in adam10-transgenic mice
we performed microarray analysis with cdna transcribed from total rna of the brains of mice aged five months.
mono-transgenic adam <dig> as well as dnadam <dig> mice were investigated in comparison to non-transgenic fvb/n wild-type mice , to analyze the influence of the α-secretase adam <dig> or its catalytically inactive form  on the gene expression profile of the cns.
to elucidate the effect of adam <dig> and dnadam <dig> on gene expression in an app background, we compared samples derived from double-transgenic adam10/app and dnadam10/app mice with those from mono-transgenic app mice.
because we wanted to test whether the modulation of adam <dig> activity might be a risk to the adult organism in respect to future therapeutic approaches, we chose  <dig> months old mice for our investigations.
at this age, app animals show cognitive deficits, whereas amyloid plaque formation occurs several months later  <cit> .
the sam plots in fig.
<dig> represent the distribution of all probe signals on the microarray chip.
depending on the statistical stringency  as represented by the red lines, significant probes are selected.
probe signals between the red lines are not significant, signals above the upper line correspond to significantly upregulated genes; signals below the lower line correspond to significantly downregulated genes.
tables  <dig> and  <dig> show the numbers of these differentially expressed genes.
adam <dig> versus fvb/n
<dig> upregulated
adam10/app versus app
<dig> upregulated
the comparison of samples from adam <dig> and fvb/n mice revealed  <dig> differentially expressed genes:  <dig> genes were up- and  <dig> genes were downregulated.
in dnadam <dig> mice, the number of regulated genes was lower; as compared to fvb/n mice,  <dig> genes were differentially expressed.
among these,  <dig> genes were up- and  <dig> genes downregulated .
against the background of app overexpression, generally more genes were found to be differentially expressed.
as compared to app mice,  <dig> genes  were differentially expressed in adam10/app mice, and more than  <dig> genes in dnadam10/app animals .
in the latter, the number of significantly regulated genes was restricted to  <dig>  including the highest up- and downregulated genes.
for the complete list of significantly regulated genes, see additional file  <dig>  tables s1-s <dig>  the data presented in this publication have been deposited in ncbi's gene expression omnibus , and are accessible by the geo series accession numbers gse <dig> and gpl <dig> .
for detection of transcripts that were commonly regulated by either adam <dig> or dnadam <dig> overexpression in mono- and double-transgenic mice, venn diagrams were generated with sam-based gene lists .
the comparison of adam <dig> versus fvb/n , and adam10/app versus app  revealed  <dig> genes which were regulated by adam <dig> overexpression in either mono- or double-transgenic mice .
when dnadam <dig> versus fvb/n  and dnadam10/app versus app, were compared, only eight genes were identified to be commonly regulated by dnadam <dig> overexpression .
this result indicates that the genetic background strongly influences the effect of adam <dig> on gene expression.
common genetic profile in mono- and double-transgenic animals
heat maps  indicate that the chips of each series had their own characteristic genetic profile.
for heat maps, genes of special interest were chosen .
the heat map in fig.
3a reveals that in mice overexpressing bovine adam <dig>  approximately the same amount of murine adam <dig> is expressed as compared to wild-type mice .
fabp <dig> is distinctly higher expressed in all dnadam <dig> mice  in contrast to wild-type mice .
the expression of nlgn <dig> in adam <dig> and dnadam <dig> mice is higher  than in fvb/n mice ).
finally, s100a <dig> and s100a <dig> show lower expression in adam <dig> and dnadam <dig> mice  in relation to fvb/n wild-type mice .
these results are in accordance with the observations made by the real-time rt-pcr as described below.
furthermore, hierarchical clustering showed that the expression profiles of the mono-transgenic mouse genes are separated to the original conditions.
in the case of heat map in fig.
3b, the small differences in the expression of mapt, gria <dig>  vldlr and lrp <dig> are fitting to the results of real-time rt-pcr analyses as described below.
hierarchical clustering revealed that the expression profiles of double-transgenic mice genes are not clearly clustered according to the experimental settings, presumably due to the more complex conditions caused by app overexpression.
also, a clear distinction between male and female mice could not be observed.
adam10-regulated biological pathways
for pathway analysis, the complete gene lists were analyzed with the bibliosphere software  and mapped to gene ontology  trees.
functional groups are only listed when their z-score of individual go-categories is higher than  <dig> .
with respect to the known cellular function of disintegrin metalloproteases in general, and the α-secretase activity of adam <dig> in particular, we investigated biological processes including cell communication , nervous system development , cell adhesion  and cell death .
furthermore, we examined neuron projection , synaptic junction  and transmission .
at the molecular level, we focused on receptor binding  and receptor activity  .
the highest number of regulated genes in adam <dig> mono-transgenic mice  belonged to the category of cell communication , followed by the categories of synaptic junction and transmission , and of nervous system development .
in dnadam <dig> mice, fewer genes were found especially in the category of cell communication .
in mono-transgenic mice, genes in the functional groups of inflammation or cell death were not over-represented .
in contrast, the category of cell death was over-represented in both double-transgenic mouse lines , probably due to app overexpression.
the major difference in the two double-transgenic lines was the 3-fold higher number of regulated genes in the category of cell communication in the adam10/app double-transgenic line , as compared to dnadam10/app mice.
the results show that overexpression of proteolytically active adam <dig> generally influences cellular communication in mice, independently of their genetic background.
one example for a regulated gene of this category is the calcium/calmodulin-dependent protein kinase ii alpha , which is upregulated in mono-transgenic adam <dig> mice  and downregulated in dnadam <dig> mice .
other genes of this category are the ldl receptor-related protein , neuroligin  and the very low density lipoprotein receptor .
adam <dig> overexpression has been shown to increase cortical synaptogenesis as revealed by immunohistochemistry  <cit> .
accordingly, here we confirmed these results on the mrna level for two neurotransmitter systems: the glutamate receptor gria <dig> and the glutamic acid decarboxylase  <dig>  as well as the gaba-a receptor subunit alpha  <dig> .
these are examples of up-regulated genes within the category of synaptic junction and transmission .
because adam <dig> has proteolytical activity, we were also interested in gene expression of putative adam <dig> substrates like app and egfr .
their expressions were not regulated in mono-transgenic mice, and therefore they are not listed in tables  <dig>   <dig>   <dig>   <dig> and tables s1-s <dig>
notch- <dig> expression was not changed in mice aged  <dig> months and its target gene hes <dig> was only slightly affected in adam <dig> mice .
however, it has been reported that the adam <dig> knock out leads to severely affected notch signaling and embryonic lethality at day  <dig>   <cit> .
as in our transgenic animals adam <dig> was under control of the postnatal active neuron-specific mouse thy 1-promoter, adam <dig> has no effect during embryogenesis.
to examine whether the lack of influence of adam <dig> on the notch pathway in our transgenic mice is due to the relative late stage  of investigation, we analyzed the expression of the notch- <dig> target gene hes <dig> in transgenic mice aged  <dig> days : about 40% induction was observed in the adam <dig> overexpressing mice and a reduction of about 50% in the dnadam <dig> transgenic mice.
in addition, we found that overexpression of adam <dig> and dnadam <dig> did not affect expression of either endogenous adam <dig> or of other putative α-secretases like adam <dig>  adam <dig> and bace <dig> in adult mice.
in general, the observed alteration of gene expression was low in all analyzed mouse lines .
alzheimer disease-related genes regulated by adam10
the genecards database , which contains  <dig> genes connected with ad , was used for identification of ad-related genes regulated by adam <dig>
in adam <dig> mice,  <dig> ad genes  were differently regulated, and in dnadam <dig> mice  <dig> ad genes   were altered including genes involved in cholesterol and lipid homeostasis, like lrp <dig>  vldlr, and fatty acid-binding protein fabp <dig>  other regulated genes code for inflammation-associated members of the s <dig> protein family .
in adam10/app mice,  <dig> ad genes , and in dnadam10/app mice,  <dig> ad genes  were altered in expression .
the relatively small number of adam10-regulated ad genes in double-transgenic mice probably reflects brain dissection at the age of five months, before plaque formation begins.
in all transgenic lines, we did not detect differences in the expression of presenilins  <dig> and  <dig>  bace <dig> was slightly upregulated  in dnadam10/app mice.
the aβ-degrading enzymes neprilysin  and insulin-degrading enzyme  were also not regulated in mono-transgenic mice.
solely, in adam10/app mice, ide was slightly down-regulated .
in order to examine an influence of sex, a separate chipinspector analysis restricted to the  <dig> most up- and downregulated genes was performed with samples from both female and male double-transgenic mice .
the gene lists of female and male double-transgenic mice were then compared to the genecards ad gene list.
the percentages of altered ad-related genes in female double-transgenic adam10/app and dnadam10/appfemale mice are similar to the numbers found in male adam10/appand dnadam10/app mice .
thus, sexual dimorphism does not cause severe differences in adam10-dependent expression of ad-related genes.
one exception was the insulin-like growth factor , which was downregulated in female dnadam10/app mice , but not in the corresponding male animals .
male adam10/app versus app
<dig> upregulated
female adam10/app versus app
<dig> upregulated
genes regulated through app overexpression
to determine the effect of app overexpression on gene regulation in transgenic mice, we compared app mice with fvb/n mice, adam10/app mice with adam <dig> mice, and dnadam10/app mice with dnadam <dig> mice.
after background adjustment and normalization with the gcrma procedure, a venn diagram of genes regulated in the transgenic mice was generated .
the overlap of the three groups represents  <dig> genes regulated by app overexpression, independent of the strain background.
this high number of genes altered by app expression demonstrates the strong influence of human app overexpression in the ad mouse model used.
ad-related genes that were regulated in double-transgenic, but not in mono-transgenic mice include microtubule-associated protein tau   and the ionotropic glutamate receptors ampa  <dig>  and ampa  <dig>  .
confirmation of microarray data
for validation of the results obtained by microarray analysis, real-time rt-pcr was applied on the original rna samples .
changes in gene expression levels of selected transcripts were normalized to the gene expression of gapdh, which was not regulated in our transgenic mouse strains.
in the microarray analyses, the calcium-binding proteins  were found to be downregulated in adam <dig> and dnadam <dig> mice.
both genes are associated with various inflammatory processes including alzheimer's disease  <cit> .
by using real-time rt-pcr, significant downregulation of s100a <dig> and s100a <dig> was confirmed .
additionally, quantification of dimers of s100a <dig> and a <dig>  by elisa revealed a slight reduction in both transgenic mouse lines  which is in accordance with the findings for mrna levels.
the decrease of about  <dig> to 15% of calprotectin as compared to wild-type mice was not statistically significant which might be due to elisa-specific detection of heterodimers.
we cannot exclude that changes concerning both monomeric proteins might be more substantial, but a detection of the monomeric form of s100a <dig> by western blotting failed as a consequence of its low expression level.
fatty acid-binding protein  <dig> , which is elevated in down syndrome fetal brains  <cit> , was found to be upregulated in dnadam <dig> mice by microarray analysis.
a significantly increased fabp <dig> expression was confirmed in dnadam <dig> mice by real-time rt-pcr.
as observed by real-time rt-pcr, expression of fabp <dig> was slightly reduced in adam <dig> mice, but this effect did not reach a significant level .
fabp <dig> protein expression was analyzed in the soluble protein fraction from brains of mono-transgenic mice by western blotting.
while adam <dig> had no significant effect on fabp <dig> expression, the dominant-negative form dnadam <dig> increased the amount of the fabp <dig> protein , which is in accordance with the results obtained by microarray and pcr analysis.
neuroligin  <dig>  a postsynaptic cell-adhesion molecule of excitatory synapses, plays a role in neuronal differentiation and axogenesis  <cit> .
in microarray analyses, neuroligin  <dig> gene expression was induced in adam <dig> mice.
this tendency, although without reaching significance, was also confirmed in real-time rt-pcr .
other proteins identified by gene profiling and associated with alzheimer disease are the low density lipoprotein receptor-related protein  <dig>   <cit> , the very low density lipoprotein receptor   <cit> , the microtubule-associated protein tau   <cit>  and the ionotropic glutamate receptors ampa <dig> and ampa <dig>   <cit> .
downregulation of lrp <dig> by adam <dig>  as observed in the chip analyses, was not confirmed by real-time rt-pcr .
for vldlr, we found by real-time rt-pcr a significant downregulation in adam10/app mice, but its upregulation in dnadam10/app mice, as detected with the microarray, could not be confirmed .
by real-time rt-pcr, the microtubule-associated protein tau was shown to be significantly downregulated in both double-transgenic mouse lines .
also in the case of the ionotropic glutamate receptors ampa <dig>  and ampa <dig> , real-time rt-pcr confirmed the results of the microarray analyses: both genes are downregulated in adam10/app mice .
discussion
increasing the α-secretase cleavage of app represents a plausible strategy for the treatment of alzheimer disease, because via this route it is possible to decrease the concentration of neurotoxic aβ peptides and to increase the amount of neuroprotective appsα simultaneously.
the aim of this study was to investigate the influence of increased amounts of adam <dig> proteins on gene expression in the mouse cns.
to this end, we analyzed transgenic mice either overexpressing catalytically active adam <dig>  or a dominant-negative mutant of adam <dig>  which is able to inhibit endogenous mouse enzymes with α-secretase activity  <cit> .
an additional reason for investigation of dnadam <dig> mice is determined by the multi-domain structure of adams because specific biological functions have been assigned to protein domains outside the catalytic centre of adams  <cit> .
in adam <dig> mice, more genes were regulated than in dnadam <dig> animals; this indicates that, due to the many substrates of adam <dig>  an increase in their cleavage products might change the expression of genes involved in cell communication and synaptic transmission.
no change, however, was detected in the expression of the substrates as a feedback reaction.
in all transgenic mice the endogenous adam <dig> level was not influenced through overexpression of adam <dig> or its inactive variant as revealed by real-time rt-pcr.
also the other adam family members adam <dig> and adam17/tace were not regulated differentially in the investigated transgenic mice, thus indicating that a reduced α-secretase activity as observed in dnadam <dig> mice  <cit>  was not compensated by the induction of gene expression of other potential α-secretases.
since adam <dig> has been implicated in notch signaling  <cit> , we investigated this pathway.
on the rna level, we found no regulation of notch- <dig> in mono- and double-transgenic mice at the age of  <dig> months: expression of the notch target gene hes <dig> was only slightly changed in mono-transgenic adam <dig> mice.
this is in accordance with earlier real-time rt-pcr experiments, where no significant difference was found in hes <dig> transcription levels between adult mice overexpressing adam <dig> and non-transgenic mice  <cit> .
this lack of influence on notch signaling is probably due to the late stage of analysis, since we found small but significant effects of adam <dig> on hes <dig> mrna levels in transgenic mice aged  <dig> days.
adam <dig> has been reported to mediate cadherin shedding, β-catenin translocation and expression of β-catenin target genes  <cit> .
in double-transgenic dnadam10/app mice various cadherins , β-catenin , several wnts  and jun kinase  were upregulated .
the upregulation of these genes might represent a compensatory mechanism to by-pass a reduced catalytic activity of adam <dig> and β-catenin signaling.
in mice overexpressing active adam <dig>  no significant changes of β-catenin target genes, for example c-myc and cyclin d <dig>  were found.
also for other adam <dig> substrates like l1cam, proteins involved in inflammation like fasl, and for growth factor receptors like egfr , we could not demonstrate any alteration.
most genes in adam <dig> and adam10/app mice were found to be altered in the pathway of cell communication, followed by genes in categories of nervous system development and synaptic junction and transmission .
one example for a regulated gene in the category of cell communication and synaptic function is the calcium/calmodulin-dependent protein kinase ii alpha , one of the most abundant kinases in the brain, which is involved in long term potentiation.
camk2α was upregulated in adam <dig> mice, and downregulated in dnadam <dig>  another gene of cell communication and synaptic function is neuroligin , a brain-specific acetylcholinesterase homologous protein, which was upregulated in adam10/app mice .
this component of excitatory synapses plays a role in neuronal differentiation and axogenesis  <cit> .
an increase in cortical synaptogenesis as found by bell et al.
in adam <dig> mice  <cit> , was confirmed through upregulation of the glutamate receptor gria <dig> and the glutamic acid decarboxylase  <dig>  as well as the gaba-a receptor subunit alpha  <dig> .
downregulation of the ionotropic glutamate receptors ampa <dig>  and ampa <dig>  as observed in our microarray study was confirmed by real-time rt-pcr: reduced mrna levels of gria <dig> and gria <dig> were detected in adam10/app mice.
the downregulation of these two genes possibly depends on overexpression of app as described before  <cit> .
the number of regulated genes involved in the development of ad was relatively small in the brains of double-transgenic adam10/app and dnadam10/app mice, and almost equivalent to mono-transgenic adam <dig> or dnadam <dig> mice .
we did not detect differences in most genes directly involved in app processing; but reduction of α-secretase activity induced a slight upregulation of bace <dig> in dnadam10/app mice.
comparative gcrma analysis demonstrated the strong influence of human app overexpression on gene expression in double-transgenic mice.
tau  was directly downregulated through app overexpression in adam10/app versus adam <dig> mice.
altered expression of ad-related genes was independent of sex, with one exception: insulin-like growth factor  <dig> , which has been implicated in alzheimer pathology  <cit> , was downregulated in double-transgenic female dnadam10/app mice.
by microarray analysis, we observed in mono-transgenic mice a downregulation of members of the s <dig> protein family, small calcium-binding proteins responsible for a wide range of intra- and extracellular functions  <cit> .
s100a <dig> and s100a <dig> were expressed to a lower extent in adam <dig> and dnadam <dig> mice.
pcr analysis and elisa confirmed this effect .
s100a <dig> and s100a <dig> form the dimer calprotectin which is a marker for inflammation  <cit> .
immunohistochemical analysis recently showed s100a <dig> in association with the neuropathological hallmarks of sporadic and familiar ad: it was found in senile plaques, in activated glia cells and in neurons with neurofibrillary tangle morphology  <cit> .
the downregulation of s100a <dig> by both adam <dig> and dnadam <dig> overexpression is probably mediated by their common domains .
a member of the fatty acid-binding proteins  was regulated by adam <dig> in mono-transgenic mice.
fabp <dig>  also named brain lipid-binding protein , is localized in the cytoplasm and in the nucleus, and is involved in the uptake, storage and/or delivery of fatty acids and retinoids into the nucleus  <cit> .
fabp <dig> is mainly expressed in radial glial cells, and is necessary for proper migration of immature neurons to cortical layers.
increased amounts of fabp <dig> in the brains of individuals with down syndrome suggest that higher concentrations of fabp <dig> contribute to brain abnormalities and mental retardation  <cit> .
we observed a significant upregulation of fabp <dig> mrna and protein in dnadam <dig> mice.
since in down syndrome patients α-secretase activity significantly decreases with age  <cit> , our results provide a connection between inhibition of α-secretase  and upregulation of fabp <dig>
CONCLUSIONS
this study shows that overexpression of adam <dig> or dnadam <dig> in the brain of adult mice does not lead to drastic alteration of gene expression.
in particular, adam <dig> or dnadam <dig> overexpression does not result in an increased expression of genes coding for pro-inflammatory or pro-apoptotic proteins.
on the contrary, overexpression of adam <dig> and its mutant even leads to a decreased amount of the inflammation marker calprotectin .
the relatively low number of genes affected by the adam <dig> modulation and the mild characteristic of altered expression levels might be related to the age of the mice we investigated.
since expression in the whole brain was analyzed, a higher change of gene expression may occur in single areas like the hippocampus.
from other reports it is evident that manipulation of adam <dig> in embryonic or early ontogenic stages could have severe side effects but therapeutic approaches concerning alzheimer's disease always will focus on adult patients.
our results in sum therefore provide evidence that, due to its effect on inflammation markers and on fabp <dig> expression, adam <dig> might have beneficial effects in addition to those that are due to its α-secretase activity.
these results further support the strategy of adam <dig> upregulation as a therapeutic approach for the treatment of ad.
abbreviations
: alzheimer disease; : amyloid precursor protein; : amyloid β-peptides; : a disintegrin and metalloproteinase  <dig>
authors' contributions
cp has carried out all molecular genetic experiments and has drafted the manuscript.
dt has performed the analyses of biological pathways and contributed to the manuscript draft; ww has coordinated the bioinformatic analysis.
ke has performed the western blot analysis, the elisa, quantification of hes5-mrna and co-drafted the manuscript; rp has participated in the design of the study and co-drafted the manuscript.
ff has conceived and coordinated the study, drafted the final version of the manuscript and given approval for its publication.
supplementary material
additional file 1
additional tables including differentially regulated genes in adam <dig> and mutant adam <dig> transgenic mice.
table s <dig> complete list of significantly regulated genes in mono-transgenic adam <dig> mice .
table s <dig> complete list of significantly regulated genes in mono-transgenic dnadam <dig> mice .
table s <dig> complete list of significantly regulated genes in double-transgenic adam10/app mice .
table s <dig> complete list of significantly regulated genes in double-transgenic dnadam10/app mice .
table s <dig> commonly regulated genes through adam <dig> overexpression in mono- and double transgenic mice  compared to adam10/app versus app .
table s <dig> commonly regulated genes through dnadam <dig> overexpression in mono- and double transgenic mice  compared to dnadam10/app versus app ).
table s <dig>  <dig> alzheimer disease genes by genecards
click here for file
acknowledgements
we thank dr. michael bonin and sven poths , and dr. florian wagner  for support of the microarray analysis.
we also thank annette roth  for performing the real-time rt-pcr.
we are grateful to dr. fred van leuven  for the app mice.
we acknowledge the financial support by the federal ministry of education and research  in the framework of the national genome research network , förderkennzeichen fkz01gs <dig>  fkz01gs <dig>  and fkz01gs <dig>  the financial support by the helmholtz association in the framework of the virtual institute of neurodegeneration and ageing and the helmholtz alliance for mental health in an ageing society, and the support by dfg  in the framework of the sfb 596: molecular mechanisms of neurodegeneration, subproject a <dig>

 accumulation of amyloid β-peptides in the brain is believed to contribute to the development of alzheimer disease. soluble oligomeric forms of aβ are neurotoxic. aβ, a 40– amino acid-comprising proteolytical fragment of the amyloid precursor protein, is released from app by sequential cleavages via β- and γ-secretases. however, the predominant route of app processing consists of successive cleavages by α- and γ-secretases. alpha-secretase attacks app inside the aβ sequence, and therefore prevents formation of neurotoxic aβ. in addition, the soluble n-terminal domain of app is released, which has neurotrophic and neuroprotective properties, and enhances ltp. in behavioral paradigms, appsα was demonstrated to improve memory in normal and amnesic mice. reduced amounts of appsα were detected in the cerebrospinal fluid of ad patients. proteinases of the adam family are main candidates for physiologically relevant α-secretases, and we demonstrated that adam has α-secretase activity in vitro and in cultured cells. adam10-deficient mice have been generated, but their early lethality at day e prevents a reliable analysis of adam10's α-secretase function in vivo, especially in neuronal cells. to investigate whether an increase in activity of putative α-secretases in vivo prevents plaque formation and cognitive deficits, we generated transgenic mice overexpressing either the α-secretase adam or the catalytically inactive adam mutant. neuronal overexpression of adam had no detrimental effects on adam single-transgenic mice: these animals exhibited normal behavioral abilities. we found that a moderate neuronal overexpression of adam in mice carrying the human app mutation increased the secretion of appsα, reduced the formation of aβ peptides, and prevented their deposition in plaques. functionally, impaired long-term potentiation and cognitive deficits were alleviated. expression of dominant-negative adam in app mice led to reduction of appsα and to enhancement of the number and size of amyloid plaques in the brain. histological analyses of mono-transgenic adam mice revealed an increase in cortical cholinergic, glutamatergic and gabaergic presynaptic bouton densities in months old mice; the cholinergic presynaptic bouton density remained elevated even during aging in adam mice. in addition to their metalloproteinase domain, adams contain a disintegrin domain as a modulator of cell-cell and cell-matrix interactions. as adam itself has been reported to be a substrate for ectodomain shedding by adam and subsequent cleavage by γ-secretase, the c-terminus of adam may represent a notch-like signaling molecule. thus, independent of the catalytic activity of adam which has been implicated in notch and β-catenin signaling, adam may also modulate gene expression via other domains. to analyze the influence of adam and its dominant-negative form on the gene expression profile of the central nervous system, we investigated adam and dnadam mice. we included in our study the double-transgenics adam10/app and dnadam10/app. since app mice show early phenotype changes, we investigated the gene expression in months old mice. methods animals animal husbandry was performed in accord with the guidelines of the german council on animal care. all mouse strains analyzed in this study have been described previously. the expression level of transgenic mature adam is 30% above endogenous levels and in dnadam mice the expression of the catalytically inactive adam mutant is sevenfold above endogenous adam. adam activity was determined in previous studies by quantitation of the app cleavage product appsα. in adam overexpressing mice the catalytic activity of adam against its substrate app was increased to about 250%. in mice overexpressing dnadam the endogenous app cleavage activity was reduced to about 25% as compared to app mice. for the first experimental series of the present study, female adam dnadam and fvb/n wild-type mice were investigated; for the second series, female and male adam10/app, dnadam10/app and app mice were compared. in each case, brains of three months old animals of each group were dissected and stored in rna-later at -80°c to prevent rna degradation. rna preparation and microarray analyses total rna from whole mouse brains was isolated by using the rneasy kit, including on-column dnase i digestion according to the manufacturer's recommendations. the quality of isolated rna was controlled by the lab-on-chip-system bioanalyser. the expression-profiling analysis for mono-transgenic mice was carried out at rzpd. samples from double-transgenic mice were analyzed at the microarray facility. in all cases, the mouse genome array containing probe sets of genes was used for mrna expression profiling. statistical analysis and gene annotations for the first series gene chip arrays and for the second series gene chip arrays were analyzed. data mining was performed by using the chipinspector analysis software, which identifies significant changes based on single probes. the corresponding transcripts were then assigned after a user-defined number of significant probes. for all analyses, a transcript coverage greater than three probes was chosen. by this strategy, annotation errors and errors caused by the existence of alternative transcripts are reduced. after total intensity normalization of each array, significantly changed genes were determined by significance analysis of microarrays, using the exhaustive comparison mode at a false discovery rate of % for double-transgenic, and % for mono-transgenic mice. for separate analysis of samples from double-transgenic female and male mice, a fdr of % was chosen. the resulting gene lists were restricted to the most strongly regulated genes. regulated genes were then analyzed with the bibliosphere software and mapped to gene ontology trees in order to identify their biological function. for identification of over-represented go terms, the bibliosphere software calculates a z-score for each term. the z-score represents the difference between observed and expected annotations, and is normalized to the standard deviation of a hypergeometric distribution. only go terms with a z-score >, which corresponds to a p-value of, have been considered. to identify transcripts which are affected by adam and dnadam overexpression in mono- and double-transgenic mice, we generated venn diagrams with sam-based gene lists. the expression profile of selected significantly regulated genes from microarrays was represented by heat maps using the r statistical software. hierarchical clustering was applied to investigate whether expression values can be separated according to experimental groups. in this study, two heat maps were generated: one compared the expression profiles of genes in adam and dnadam mono-transgenic mice, as well as in fvb/n non-transgenic control mice; a second one compared the expression profiles of double-transgenics and app mice. because the two series of expression arrays were measured in different laboratories, a global normalization procedure was needed to make them comparable. the default background noise adjustment, provided by the affymetrix system, is based on the difference of perfect matching probes minus mismatching probes. due to unspecific binding, the global background adjustment method robust multi-array average expression measure, which ignores the mm intensities, has been developed. because rma adjustment does not completely remove unspecific intensities, an enhanced method denoted genechip rma has been designed which considers the sequence of probes. we performed background adjustment as well as quantile normalization for all data sets with the gcrma method by using the carmaweb interface. subsequently, an unpaired two-tailed student's t-test was applied for each respective gene to determine whether it is differentially expressed in the two sample groups. since microarray analysis operates with large numbers of multiple comparisons, a false discovery rate-controlling method has to be applied. therefore, by using the benjamini-hochberg method, adjusted p-values were calculated. the gcrma method is also appropriate for detection of minor changes in gene expression, and was required for comparative analysis of mono- and double-transgenic mice, due to the low intensities of the microarrays from the first series as compared to those of the second series. by comparing data derived from mono- and double-transgenic mice, we analyzed global biological trends of adam and dnadam overexpression in fvb/n and fvb/n app strain backgrounds. to identify transcripts which were commonly affected by appoverexpression in all double-transgenic mice, we generated a venn diagram with gcrma-based gene lists. quantitative real-time rt-pcr a two-step real-time reverse transcription -pcr was used to measure the expression of candidate genes. isolated total rna was used to synthesize cdna in a μl reaction with the quantitect reverse transcription kit according to the manufacturer's manual. by adding water, the reaction volume was subsequently increased to μl. real-time rt-pcr was carried out in 96-well plates, using the abi prism sequence detection system and quantitect primer assays. the primers for selected candidate genes are listed in table real-time rt-pcr reactions in a volume of μl were performed in duplicate or triplicate under the following conditions: μl of diluted cdna, μl 2× quantitect pcr master mix and nm of respective primer pair. after the initial denaturing and enzyme activation step, cycles were performed. a single dna melting profile was observed in dissociation assay conditions demonstrating amplification of a unique product free of primer dimers. for detection of hes in day old mice a one step real-time rt-pcr was performed using the quantitect-sybr-green one-step-rt pcr-kit and ng rna in a reaction volume of μl. the specific primer pair was as follows: hes5rt_for 5'gaaaaaccgactgcggaagcc3' and hes5rt_rev 5'acggccatctccaggatgtc3'. for data analysis, the threshold cycle which indicates the relative abundance of a particular transcript, was calculated. standard curves were generated by amplification of serially diluted cdna. according to this method, the amount of all relevant genes was normalized to the amount of endogenous gapdh present in the same sample. measured values from control samples were set to 100%. changes in gene expression are presented as the mean of alteration ± sd. the data were analyzed for statistical significance using one-way anova. western blotting mouse brain tissue was stored on dry ice immediately after dissection. ice-cold tris buffer containing proteinase inhibitors was added, and tissue was homogenized in a tissue lyser. the supernatants resulting from centrifugation at rpm for hours were separated on 14% sds-gels and transferred to nitrocellulose membrane by tank blot system. for the detection and quantification of soluble fabp antibody ab, and the appropriate horseradish peroxidase-coupled secondary antibody were used. elisa hemispheres of mouse brain were weighed, proteins extracted and calprotectin was quantified as recommended by the elisa manufacturer. in brief, tissue was homogenized in extraction buffer for min at hz in a tissue lyzer and extraction was performed for min at 4°c under agitation. after centrifugation the supernatant and protein standards were added to microtiter plates in a total volume of μl in duplicates. incubation of the plate and measurement of optical densities at nm were performed following the manufacturer's instructions. the relative amount of calprotectin was calculated by division of background-corrected values by wet tissue weight.  microarray analysis of gene regulation in adam10-transgenic mice we performed microarray analysis with cdna transcribed from total rna of the brains of mice aged five months. mono-transgenic adam as well as dnadam mice were investigated in comparison to non-transgenic fvb/n wild-type mice, to analyze the influence of the α-secretase adam or its catalytically inactive form on the gene expression profile of the cns. to elucidate the effect of adam and dnadam on gene expression in an app background, we compared samples derived from double-transgenic adam10/app and dnadam10/app mice with those from mono-transgenic app mice. because we wanted to test whether the modulation of adam activity might be a risk to the adult organism in respect to future therapeutic approaches, we chose months old mice for our investigations. at this age, app animals show cognitive deficits, whereas amyloid plaque formation occurs several months later. the sam plots in fig. represent the distribution of all probe signals on the microarray chip. depending on the statistical stringency as represented by the red lines, significant probes are selected. probe signals between the red lines are not significant, signals above the upper line correspond to significantly upregulated genes; signals below the lower line correspond to significantly downregulated genes. tables and show the numbers of these differentially expressed genes. adam versus fvb/n upregulated adam10/app versus app upregulated the comparison of samples from adam and fvb/n mice revealed differentially expressed genes: genes were up- and genes were downregulated. in dnadam mice, the number of regulated genes was lower; as compared to fvb/n mice, genes were differentially expressed. among these, genes were up- and genes downregulated. against the background of app overexpression, generally more genes were found to be differentially expressed. as compared to app mice, genes were differentially expressed in adam10/app mice, and more than genes in dnadam10/app animals. in the latter, the number of significantly regulated genes was restricted to including the highest up- and downregulated genes. for the complete list of significantly regulated genes, see additional file tables s1-s the data presented in this publication have been deposited in ncbi's gene expression omnibus, and are accessible by the geo series accession numbers gse and gpl. for detection of transcripts that were commonly regulated by either adam or dnadam overexpression in mono- and double-transgenic mice, venn diagrams were generated with sam-based gene lists. the comparison of adam versus fvb/n, and adam10/app versus app revealed genes which were regulated by adam overexpression in either mono- or double-transgenic mice. when dnadam versus fvb/n and dnadam10/app versus app, were compared, only eight genes were identified to be commonly regulated by dnadam overexpression. this result indicates that the genetic background strongly influences the effect of adam on gene expression. common genetic profile in mono- and double-transgenic animals heat maps indicate that the chips of each series had their own characteristic genetic profile. for heat maps, genes of special interest were chosen. the heat map in fig. 3a reveals that in mice overexpressing bovine adam approximately the same amount of murine adam is expressed as compared to wild-type mice. fabp is distinctly higher expressed in all dnadam mice in contrast to wild-type mice. the expression of nlgn in adam and dnadam mice is higher than in fvb/n mice ). finally, s100a and s100a show lower expression in adam and dnadam mice in relation to fvb/n wild-type mice. these results are in accordance with the observations made by the real-time rt-pcr as described below. furthermore, hierarchical clustering showed that the expression profiles of the mono-transgenic mouse genes are separated to the original conditions. in the case of heat map in fig. 3b, the small differences in the expression of mapt, gria vldlr and lrp are fitting to the results of real-time rt-pcr analyses as described below. hierarchical clustering revealed that the expression profiles of double-transgenic mice genes are not clearly clustered according to the experimental settings, presumably due to the more complex conditions caused by app overexpression. also, a clear distinction between male and female mice could not be observed. adam10-regulated biological pathways for pathway analysis, the complete gene lists were analyzed with the bibliosphere software and mapped to gene ontology trees. functional groups are only listed when their z-score of individual go-categories is higher than. with respect to the known cellular function of disintegrin metalloproteases in general, and the α-secretase activity of adam in particular, we investigated biological processes including cell communication, nervous system development, cell adhesion and cell death. furthermore, we examined neuron projection, synaptic junction and transmission. at the molecular level, we focused on receptor binding and receptor activity. the highest number of regulated genes in adam mono-transgenic mice belonged to the category of cell communication, followed by the categories of synaptic junction and transmission, and of nervous system development. in dnadam mice, fewer genes were found especially in the category of cell communication. in mono-transgenic mice, genes in the functional groups of inflammation or cell death were not over-represented. in contrast, the category of cell death was over-represented in both double-transgenic mouse lines, probably due to app overexpression. the major difference in the two double-transgenic lines was the 3-fold higher number of regulated genes in the category of cell communication in the adam10/app double-transgenic line, as compared to dnadam10/app mice. the results show that overexpression of proteolytically active adam generally influences cellular communication in mice, independently of their genetic background. one example for a regulated gene of this category is the calcium/calmodulin-dependent protein kinase ii alpha, which is upregulated in mono-transgenic adam mice and downregulated in dnadam mice. other genes of this category are the ldl receptor-related protein, neuroligin and the very low density lipoprotein receptor. adam overexpression has been shown to increase cortical synaptogenesis as revealed by immunohistochemistry. accordingly, here we confirmed these results on the mrna level for two neurotransmitter systems: the glutamate receptor gria and the glutamic acid decarboxylase as well as the gaba-a receptor subunit alpha. these are examples of up-regulated genes within the category of synaptic junction and transmission. because adam has proteolytical activity, we were also interested in gene expression of putative adam substrates like app and egfr. their expressions were not regulated in mono-transgenic mice, and therefore they are not listed in tables and tables s1-s notch- expression was not changed in mice aged months and its target gene hes was only slightly affected in adam mice. however, it has been reported that the adam knock out leads to severely affected notch signaling and embryonic lethality at day. as in our transgenic animals adam was under control of the postnatal active neuron-specific mouse thy 1-promoter, adam has no effect during embryogenesis. to examine whether the lack of influence of adam on the notch pathway in our transgenic mice is due to the relative late stage of investigation, we analyzed the expression of the notch- target gene hes in transgenic mice aged days: about 40% induction was observed in the adam overexpressing mice and a reduction of about 50% in the dnadam transgenic mice. in addition, we found that overexpression of adam and dnadam did not affect expression of either endogenous adam or of other putative α-secretases like adam adam and bace in adult mice. in general, the observed alteration of gene expression was low in all analyzed mouse lines. alzheimer disease-related genes regulated by adam10 the genecards database, which contains genes connected with ad, was used for identification of ad-related genes regulated by adam in adam mice, ad genes were differently regulated, and in dnadam mice ad genes were altered including genes involved in cholesterol and lipid homeostasis, like lrp vldlr, and fatty acid-binding protein fabp other regulated genes code for inflammation-associated members of the s protein family. in adam10/app mice, ad genes, and in dnadam10/app mice, ad genes were altered in expression. the relatively small number of adam10-regulated ad genes in double-transgenic mice probably reflects brain dissection at the age of five months, before plaque formation begins. in all transgenic lines, we did not detect differences in the expression of presenilins and bace was slightly upregulated in dnadam10/app mice. the aβ-degrading enzymes neprilysin and insulin-degrading enzyme were also not regulated in mono-transgenic mice. solely, in adam10/app mice, ide was slightly down-regulated. in order to examine an influence of sex, a separate chipinspector analysis restricted to the most up- and downregulated genes was performed with samples from both female and male double-transgenic mice. the gene lists of female and male double-transgenic mice were then compared to the genecards ad gene list. the percentages of altered ad-related genes in female double-transgenic adam10/app and dnadam10/appfemale mice are similar to the numbers found in male adam10/appand dnadam10/app mice. thus, sexual dimorphism does not cause severe differences in adam10-dependent expression of ad-related genes. one exception was the insulin-like growth factor, which was downregulated in female dnadam10/app mice, but not in the corresponding male animals. male adam10/app versus app upregulated female adam10/app versus app upregulated genes regulated through app overexpression to determine the effect of app overexpression on gene regulation in transgenic mice, we compared app mice with fvb/n mice, adam10/app mice with adam mice, and dnadam10/app mice with dnadam mice. after background adjustment and normalization with the gcrma procedure, a venn diagram of genes regulated in the transgenic mice was generated. the overlap of the three groups represents genes regulated by app overexpression, independent of the strain background. this high number of genes altered by app expression demonstrates the strong influence of human app overexpression in the ad mouse model used. ad-related genes that were regulated in double-transgenic, but not in mono-transgenic mice include microtubule-associated protein tau and the ionotropic glutamate receptors ampa and ampa. confirmation of microarray data for validation of the results obtained by microarray analysis, real-time rt-pcr was applied on the original rna samples. changes in gene expression levels of selected transcripts were normalized to the gene expression of gapdh, which was not regulated in our transgenic mouse strains. in the microarray analyses, the calcium-binding proteins were found to be downregulated in adam and dnadam mice. both genes are associated with various inflammatory processes including alzheimer's disease. by using real-time rt-pcr, significant downregulation of s100a and s100a was confirmed. additionally, quantification of dimers of s100a and a by elisa revealed a slight reduction in both transgenic mouse lines which is in accordance with the findings for mrna levels. the decrease of about to 15% of calprotectin as compared to wild-type mice was not statistically significant which might be due to elisa-specific detection of heterodimers. we cannot exclude that changes concerning both monomeric proteins might be more substantial, but a detection of the monomeric form of s100a by western blotting failed as a consequence of its low expression level. fatty acid-binding protein, which is elevated in down syndrome fetal brains, was found to be upregulated in dnadam mice by microarray analysis. a significantly increased fabp expression was confirmed in dnadam mice by real-time rt-pcr. as observed by real-time rt-pcr, expression of fabp was slightly reduced in adam mice, but this effect did not reach a significant level. fabp protein expression was analyzed in the soluble protein fraction from brains of mono-transgenic mice by western blotting. while adam had no significant effect on fabp expression, the dominant-negative form dnadam increased the amount of the fabp protein, which is in accordance with the results obtained by microarray and pcr analysis. neuroligin a postsynaptic cell-adhesion molecule of excitatory synapses, plays a role in neuronal differentiation and axogenesis. in microarray analyses, neuroligin gene expression was induced in adam mice. this tendency, although without reaching significance, was also confirmed in real-time rt-pcr. other proteins identified by gene profiling and associated with alzheimer disease are the low density lipoprotein receptor-related protein, the very low density lipoprotein receptor, the microtubule-associated protein tau and the ionotropic glutamate receptors ampa and ampa. downregulation of lrp by adam as observed in the chip analyses, was not confirmed by real-time rt-pcr. for vldlr, we found by real-time rt-pcr a significant downregulation in adam10/app mice, but its upregulation in dnadam10/app mice, as detected with the microarray, could not be confirmed. by real-time rt-pcr, the microtubule-associated protein tau was shown to be significantly downregulated in both double-transgenic mouse lines. also in the case of the ionotropic glutamate receptors ampa and ampa, real-time rt-pcr confirmed the results of the microarray analyses: both genes are downregulated in adam10/app mice. discussion increasing the α-secretase cleavage of app represents a plausible strategy for the treatment of alzheimer disease, because via this route it is possible to decrease the concentration of neurotoxic aβ peptides and to increase the amount of neuroprotective appsα simultaneously. the aim of this study was to investigate the influence of increased amounts of adam proteins on gene expression in the mouse cns. to this end, we analyzed transgenic mice either overexpressing catalytically active adam or a dominant-negative mutant of adam which is able to inhibit endogenous mouse enzymes with α-secretase activity. an additional reason for investigation of dnadam mice is determined by the multi-domain structure of adams because specific biological functions have been assigned to protein domains outside the catalytic centre of adams. in adam mice, more genes were regulated than in dnadam animals; this indicates that, due to the many substrates of adam an increase in their cleavage products might change the expression of genes involved in cell communication and synaptic transmission. no change, however, was detected in the expression of the substrates as a feedback reaction. in all transgenic mice the endogenous adam level was not influenced through overexpression of adam or its inactive variant as revealed by real-time rt-pcr. also the other adam family members adam and adam17/tace were not regulated differentially in the investigated transgenic mice, thus indicating that a reduced α-secretase activity as observed in dnadam mice was not compensated by the induction of gene expression of other potential α-secretases. since adam has been implicated in notch signaling, we investigated this pathway. on the rna level, we found no regulation of notch- in mono- and double-transgenic mice at the age of months: expression of the notch target gene hes was only slightly changed in mono-transgenic adam mice. this is in accordance with earlier real-time rt-pcr experiments, where no significant difference was found in hes transcription levels between adult mice overexpressing adam and non-transgenic mice. this lack of influence on notch signaling is probably due to the late stage of analysis, since we found small but significant effects of adam on hes mrna levels in transgenic mice aged days. adam has been reported to mediate cadherin shedding, β-catenin translocation and expression of β-catenin target genes. in double-transgenic dnadam10/app mice various cadherins, β-catenin, several wnts and jun kinase were upregulated. the upregulation of these genes might represent a compensatory mechanism to by-pass a reduced catalytic activity of adam and β-catenin signaling. in mice overexpressing active adam no significant changes of β-catenin target genes, for example c-myc and cyclin d were found. also for other adam substrates like l1cam, proteins involved in inflammation like fasl, and for growth factor receptors like egfr, we could not demonstrate any alteration. most genes in adam and adam10/app mice were found to be altered in the pathway of cell communication, followed by genes in categories of nervous system development and synaptic junction and transmission. one example for a regulated gene in the category of cell communication and synaptic function is the calcium/calmodulin-dependent protein kinase ii alpha, one of the most abundant kinases in the brain, which is involved in long term potentiation. camk2α was upregulated in adam mice, and downregulated in dnadam another gene of cell communication and synaptic function is neuroligin, a brain-specific acetylcholinesterase homologous protein, which was upregulated in adam10/app mice. this component of excitatory synapses plays a role in neuronal differentiation and axogenesis. an increase in cortical synaptogenesis as found by bell et al. in adam mice, was confirmed through upregulation of the glutamate receptor gria and the glutamic acid decarboxylase as well as the gaba-a receptor subunit alpha. downregulation of the ionotropic glutamate receptors ampa and ampa as observed in our microarray study was confirmed by real-time rt-pcr: reduced mrna levels of gria and gria were detected in adam10/app mice. the downregulation of these two genes possibly depends on overexpression of app as described before. the number of regulated genes involved in the development of ad was relatively small in the brains of double-transgenic adam10/app and dnadam10/app mice, and almost equivalent to mono-transgenic adam or dnadam mice. we did not detect differences in most genes directly involved in app processing; but reduction of α-secretase activity induced a slight upregulation of bace in dnadam10/app mice. comparative gcrma analysis demonstrated the strong influence of human app overexpression on gene expression in double-transgenic mice. tau was directly downregulated through app overexpression in adam10/app versus adam mice. altered expression of ad-related genes was independent of sex, with one exception: insulin-like growth factor, which has been implicated in alzheimer pathology, was downregulated in double-transgenic female dnadam10/app mice. by microarray analysis, we observed in mono-transgenic mice a downregulation of members of the s protein family, small calcium-binding proteins responsible for a wide range of intra- and extracellular functions. s100a and s100a were expressed to a lower extent in adam and dnadam mice. pcr analysis and elisa confirmed this effect. s100a and s100a form the dimer calprotectin which is a marker for inflammation. immunohistochemical analysis recently showed s100a in association with the neuropathological hallmarks of sporadic and familiar ad: it was found in senile plaques, in activated glia cells and in neurons with neurofibrillary tangle morphology. the downregulation of s100a by both adam and dnadam overexpression is probably mediated by their common domains. a member of the fatty acid-binding proteins was regulated by adam in mono-transgenic mice. fabp also named brain lipid-binding protein, is localized in the cytoplasm and in the nucleus, and is involved in the uptake, storage and/or delivery of fatty acids and retinoids into the nucleus. fabp is mainly expressed in radial glial cells, and is necessary for proper migration of immature neurons to cortical layers. increased amounts of fabp in the brains of individuals with down syndrome suggest that higher concentrations of fabp contribute to brain abnormalities and mental retardation. we observed a significant upregulation of fabp mrna and protein in dnadam mice. since in down syndrome patients α-secretase activity significantly decreases with age, our results provide a connection between inhibition of α-secretase and upregulation of fabp  this study shows that overexpression of adam or dnadam in the brain of adult mice does not lead to drastic alteration of gene expression. in particular, adam or dnadam overexpression does not result in an increased expression of genes coding for pro-inflammatory or pro-apoptotic proteins. on the contrary, overexpression of adam and its mutant even leads to a decreased amount of the inflammation marker calprotectin. the relatively low number of genes affected by the adam modulation and the mild characteristic of altered expression levels might be related to the age of the mice we investigated. since expression in the whole brain was analyzed, a higher change of gene expression may occur in single areas like the hippocampus. from other reports it is evident that manipulation of adam in embryonic or early ontogenic stages could have severe side effects but therapeutic approaches concerning alzheimer's disease always will focus on adult patients. our results in sum therefore provide evidence that, due to its effect on inflammation markers and on fabp expression, adam might have beneficial effects in addition to those that are due to its α-secretase activity. these results further support the strategy of adam upregulation as a therapeutic approach for the treatment of ad. abbreviations: alzheimer disease;: amyloid precursor protein;: amyloid β-peptides;: a disintegrin and metalloproteinase authors' contributions cp has carried out all molecular genetic experiments and has drafted the manuscript. dt has performed the analyses of biological pathways and contributed to the manuscript draft; ww has coordinated the bioinformatic analysis. ke has performed the western blot analysis, the elisa, quantification of hes5-mrna and co-drafted the manuscript; rp has participated in the design of the study and co-drafted the manuscript. ff has conceived and coordinated the study, drafted the final version of the manuscript and given approval for its publication. supplementary material additional file 1 additional tables including differentially regulated genes in adam and mutant adam transgenic mice. table s complete list of significantly regulated genes in mono-transgenic adam mice. table s complete list of significantly regulated genes in mono-transgenic dnadam mice. table s complete list of significantly regulated genes in double-transgenic adam10/app mice. table s complete list of significantly regulated genes in double-transgenic dnadam10/app mice. table s commonly regulated genes through adam overexpression in mono- and double transgenic mice compared to adam10/app versus app. table s commonly regulated genes through dnadam overexpression in mono- and double transgenic mice compared to dnadam10/app versus app ). table s alzheimer disease genes by genecards click here for file acknowledgements we thank dr. michael bonin and sven poths, and dr. florian wagner for support of the microarray analysis. we also thank annette roth for performing the real-time rt-pcr. we are grateful to dr. fred van leuven for the app mice. we acknowledge the financial support by the federal ministry of education and research in the framework of the national genome research network, förderkennzeichen fkz01gs fkz01gs and fkz01gs the financial support by the helmholtz association in the framework of the virtual institute of neurodegeneration and ageing and the helmholtz alliance for mental health in an ageing society, and the support by dfg in the framework of the sfb 596: molecular mechanisms of neurodegeneration, subproject a 
overexpression of proteolytically active adam affected several physiological pathways, such as cell communication, nervous system development, neuron projection as well as synaptic transmission. these results suggest that upregulation of adam in the brain has beneficial effects on ad pathology. these results are further supportive for the strategy to treat ad by increasing the α-secretase activity. in a transgenic mouse model of alzheimer disease, cleavage of the amyloid precursor protein by the α-secretase adam prevented amyloid plaque formation, and alleviated cognitive deficits. to assess the influence of adam on the gene expression profile in the brain, we performed a microarray analysis using rna isolated from brains of five months old mice overexpressing either the α-secretase adam or a dominant-negative mutant of this enzyme. although adam has been implicated in notch and β-catenin signaling, no significant changes in the respective target genes were observed in adult adam transgenic mice. in general, there was only a moderate alteration of gene expression in adam overexpressing mice. even a decrease of inflammation markers was observed. real-time rt-pcr confirmed a downregulation of genes coding for the inflammation-associated proteins s100a and s100a induced by moderate adam overexpression. genes coding for pro-inflammatory or pro-apoptotic proteins were not over-represented among differentially regulated genes. furthermore, adam overexpression increased the cortical synaptogenesis.
 in a transgenic mouse model of alzheimer disease, cleavage of the amyloid precursor protein by the α-secretase adam prevented amyloid plaque formation, and alleviated cognitive deficits. furthermore, adam overexpression increased the cortical synaptogenesis. these results suggest that upregulation of adam in the brain has beneficial effects on ad pathology.  to assess the influence of adam on the gene expression profile in the brain, we performed a microarray analysis using rna isolated from brains of five months old mice overexpressing either the α-secretase adam or a dominant-negative mutant of this enzyme. as compared to non-transgenic wild-type mice, in adam transgenic mice genes, and in dnadam mice genes were found to be differentially expressed. a higher number of genes was differentially regulated in double-transgenic mouse strains additionally expressing the human app mutant. overexpression of proteolytically active adam affected several physiological pathways, such as cell communication, nervous system development, neuron projection as well as synaptic transmission. although adam has been implicated in notch and β-catenin signaling, no significant changes in the respective target genes were observed in adult adam transgenic mice. real-time rt-pcr confirmed a downregulation of genes coding for the inflammation-associated proteins s100a and s100a induced by moderate adam overexpression. overexpression of the dominant-negative form dnadam led to a significant increase in the expression of the fatty acid-binding protein fabp which also has been found in higher amounts in brains of down syndrome patients.  in general, there was only a moderate alteration of gene expression in adam overexpressing mice. genes coding for pro-inflammatory or pro-apoptotic proteins were not over-represented among differentially regulated genes. even a decrease of inflammation markers was observed. these results are further supportive for the strategy to treat ad by increasing the α-secretase activity. 