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 ADAM10 proteins on gene expression in the mouse CNS. To this end, we analyzed transgenic mice either overexpressing catalytically active ADAM10, or a dominant-negative mutant of ADAM10 (dnADAM10) which is able to inhibit endogenous mouse enzymes with α-secretase activity [10
]. An additional reason for investigation of dnADAM10 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 [15
In ADAM10 mice, more genes were regulated than in dnADAM10 animals; this indicates that, due to the many substrates of ADAM10, 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 ADAM10 level was not influenced through overexpression of ADAM10 or its inactive variant as revealed by real-time RT-PCR. Also the other ADAM family members Adam9 and Adam17/TACE were not regulated differentially in the investigated transgenic mice, thus indicating that a reduced α-secretase activity as observed in dnADAM10 mice [12
] was not compensated by the induction of gene expression of other potential α-secretases.
Since ADAM10 has been implicated in Notch signaling [11
], we investigated this pathway. On the RNA level, we found no regulation of Notch-1 in mono- and double-transgenic mice at the age of 5 months: expression of the Notch target gene Hes5 was only slightly changed in mono-transgenic ADAM10 mice. This is in accordance with earlier real-time RT-PCR experiments, where no significant difference was found in Hes5 transcription levels between adult mice overexpressing ADAM10 and non-transgenic mice [12
]. This lack of influence on Notch signaling is probably due to the late stage of analysis, since we found small but significant effects of ADAM10 on Hes5 mRNA levels in transgenic mice aged 15 days.
ADAM10 has been reported to mediate cadherin shedding, β-catenin translocation and expression of β-catenin target genes [33
]. In double-transgenic dnADAM10/APP[V717I]
mice various cadherins (Cdh8, Cdh10 and Cdh13), β-catenin (Ctnnb1), several Wnts (Wnt4, Wnt7a and Wnt9a) and Jun kinase (Jun) were upregulated (about 30%). The upregulation of these genes might represent a compensatory mechanism to by-pass a reduced catalytic activity of ADAM10 and β-catenin signaling. In mice overexpressing active ADAM10, no significant changes of β-catenin target genes, for example c-myc and cyclin D1, were found.
Also for other ADAM10 substrates like L1cam, proteins involved in inflammation like Fasl, and for growth factor receptors like Egfr (see also table ), we could not demonstrate any alteration.
Most genes in ADAM10 and ADAM10/APP[V717I]
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 (Tab. , , , ). One example for a regulated gene in the category of cell communication and synaptic function is the calcium/calmodulin-dependent protein kinase II alpha (Camk2α), one of the most abundant kinases in the brain, which is involved in long term potentiation. Camk2α was upregulated in ADAM10 mice, and downregulated in dnADAM10. Another gene of cell communication and synaptic function is neuroligin (Nlgn1), a brain-specific acetylcholinesterase homologous protein, which was upregulated in ADAM10/APP[V717I]
mice (Fig. ). This component of excitatory synapses plays a role in neuronal differentiation and axogenesis [27
]. An increase in cortical synaptogenesis as found by Bell et al. in ADAM10 mice [14
], was confirmed through upregulation of the glutamate receptor Gria3 and the glutamic acid decarboxylase 2 (Gad2) as well as the GABA-A receptor subunit alpha 4 (Gabra4).
Downregulation of the ionotropic glutamate receptors AMPA1 (Gria1) and AMPA2 (Gria2) as observed in our microarray study was confirmed by real-time RT-PCR: reduced mRNA levels of Gria1 and Gria2 were detected in ADAM10/APP[V717I]
mice. The downregulation of these two genes possibly depends on overexpression of APP[V717I]
as described before [35
The number of regulated genes involved in the development of AD was relatively small in the brains of double-transgenic ADAM10/APP[V717I] and dnADAM10/APP[V717I] mice, and almost equivalent to mono-transgenic ADAM10 or dnADAM10 mice (Fig. ). We did not detect differences in most genes directly involved in APP processing; but reduction of α-secretase activity induced a slight upregulation of Bace1 in dnADAM10/APP[V717I] mice.
Comparative GCRMA analysis demonstrated the strong influence of human APP[V717I]
overexpression on gene expression in double-transgenic mice. Tau (Mapt) was directly downregulated through APP[V717I]
overexpression in ADAM10/APP[V717I]
versus ADAM10 mice. Altered expression of AD-related genes was independent of sex, with one exception: insulin-like growth factor 1 (Igf-1), which has been implicated in Alzheimer pathology [37
], was downregulated in double-transgenic female dnADAM10/APP[V717I]
By microarray analysis, we observed in mono-transgenic mice a downregulation of members of the S100 protein family, small calcium-binding proteins responsible for a wide range of intra- and extracellular functions [39
]. S100a8 and S100a9 were expressed to a lower extent in ADAM10 and dnADAM10 mice. PCR analysis and ELISA confirmed this effect (Fig. and , Figure ). S100a8 and S100a9 form the dimer calprotectin which is a marker for inflammation [40
]. Immunohistochemical analysis recently showed S100A9 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 [25
]. The downregulation of S100a9 by both ADAM10 and dnADAM10 overexpression is probably mediated by their common domains (the disintegrin and cystein-rich domain as well as the C-terminus).
A member of the fatty acid-binding proteins (Fabp7) was regulated by ADAM10 in mono-transgenic mice. Fabp7, also named brain lipid-binding protein (B-Fabp), 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 [41
]. Fabp7 is mainly expressed in radial glial cells, and is necessary for proper migration of immature neurons to cortical layers. Increased amounts of Fabp7 in the brains of individuals with Down syndrome suggest that higher concentrations of Fabp7 contribute to brain abnormalities and mental retardation [26
]. We observed a significant upregulation of Fabp7 mRNA and protein in dnADAM10 mice. Since in Down syndrome patients α-secretase activity significantly decreases with age [42
], our results provide a connection between inhibition of α-secretase (in our study by dnADAM10) and upregulation of Fabp7.