The Amyloid Precursor Protein (APP) Triplicated Gene Impairs Neuronal Precursor Differentiation and Neurite Development through Two Different Domains in the Ts65Dn Mouse Model for Down Syndrome*

Background: Individuals with Down syndrome suffer from mental retardation due to severe neurogenesis impairment. Results: Normalization of the triplicated gene APP expression restores neuronal maturation and differentiation in trisomic neuronal precursors. Conclusion: APP overproduction contributes to neurogenesis impairment in DS. Significance: APP signaling may be a target for therapeutic approaches aiming to improve brain development in DS. Intellectual disability in Down syndrome (DS) appears to be related to severe proliferation impairment during brain development. Recent evidence shows that it is not only cellular proliferation that is heavily compromised in DS, but also cell fate specification and dendritic maturation. The amyloid precursor protein (APP), a gene that is triplicated in DS, plays a key role in normal brain development by influencing neural precursor cell proliferation, cell fate specification, and neuronal maturation. APP influences these processes via two separate domains, the APP intracellular domain (AICD) and the soluble secreted APP. We recently found that the proliferation impairment of neuronal precursors (NPCs) from the Ts65Dn mouse model for DS was caused by derangement of the Shh pathway due to overexpression of patched1(Ptch1), its inhibitory regulator. Ptch1 overexpression was related to increased levels within the APP/AICD system. The overall goal of this study was to determine whether APP contributes to neurogenesis impairment in DS by influencing in addition to proliferation, cell fate specification, and neurite development. We found that normalization of APP expression restored the reduced neuronogenesis, the increased astrogliogenesis, and the reduced neurite length of trisomic NPCs, indicating that APP overexpression underpins all aspects of neurogenesis impairment. Moreover, we found that two different domains of APP impair neuronal differentiation and maturation in trisomic NPCs. The APP/AICD system regulates neuronogenesis and neurite length through the Shh pathway, whereas the APP/secreted AP system promotes astrogliogenesis through an IL-6-associated signaling cascade. These results provide novel insight into the mechanisms underlying brain development alterations in DS.

Down syndrome (DS) 2 is a genetic pathology caused by triplication of human chromosome 21. Individuals with DS may have various medical problems, but intellectual disability is the unavoidable hallmark and the most invalidating aspect of this pathology with a notable impact on public health (1,2). The major determinant of intellectual disability is considered to be the characteristically decreased brain size of individuals with DS. The hypocellularity observed in the primary visual cortex, primary somatosensory cortex, primary motor cortex, primary auditory cortex, and superior temporal gyrus of individuals with DS led to the hypothesis that proliferation deficits may underlie the typical hypocellularity of the DS brain (3,4). Consistently with this idea, recent evidence has shown a severe impairment of cellular proliferation in the ventricular germinal matrix and various structures of the hippocampal region and cerebellum of human fetuses with DS (5)(6)(7). This proliferation impairment is worsened by impaired cell fate specification with a reduction in neuronogenesis and an increase in astrogliogenesis (5)(6)(7). This evidence suggests that proliferation impairment and cell fate specification may be key determinants of intellectual disability in individuals with DS. Dendritic pathology is also a consistent feature and a possible substrate for mental retardation in DS. In children and adults with DS, there is a marked reduction in dendritic branching and spine density (8 -12). The Ts65Dn mouse model of DS is one of the models that is most comparable to human DS both in terms of triplicated genes and phenotype (13). Similar to individuals with DS, the Ts65Dn mouse shows severe neurogenesis impairment in numerous brain regions (5, 14 -17) and less branched and less spinous dendrites (18,19).
The role played by different triplicated genes in brain developmental alterations in DS is scarcely elucidated. Recent findings demonstrate that the amyloid precursor protein (APP) plays a key role in normal brain development by influencing neural precursor cell proliferation, cell fate specification, and maturation (20), which suggests that triplication of this gene may compromise these processes in the DS brain. APP influences cell fate specification and neuronal maturation via two separate domains, the soluble secreted APP (sAPP) and the APP intracellular domain (AICD). sAPP promotes gliogenesis (20 -22), whereas AICD negatively modulates proliferation and maturation of neural precursors (20). APP is cleaved by three types of proteases, which are designated ␣-, ␤-, and ␥-secretases and which may result in functionally distinct outcomes. The non-amyloidogenic APP processing pathway involves proteolytic cleavages exerted by ␣and ␥-secretases resulting in the generation of N-terminal sAPP␣ and C-terminal fragments including P3, CTF83, and the intracellular domain (AICD). The alternative amyloidogenic APP processing pathway involves proteolytic cleavages exerted by ␤and ␥-secretases resulting in the generation of sAPP␤ and C-terminal fragments including A␤, CTF99, and AICD.
AICD has been shown to be involved in the transcriptional regulation of various genes (23,24), including patched1 (Ptch1) (25). Recently, we demonstrated that neuronal precursors from the Ts65Dn mouse exhibit derangement of the Sonic Hedgehog (Shh) pathway due to an overexpression of Ptch1, its inhibitory regulator (26). We found that Ptch1 overexpression was correlated to increased levels of AICD (26), indicating that the APP/ AICD system may underlie derangement of Shh signaling, and consequently, the proliferation defects that characterize the DS brain.
Shh signaling appears to regulate not only neural progenitor cell division but also cell fate specification and neuronal maturation (27), suggesting that the APP/AICD-dependent derangement of the Shh pathway in the DS brain may underlie, in addition to proliferation impairment, defective cell fate specification and neuronal maturation. Moreover, increased levels of the sAPP fragment may also contribute to derangement of these processes (20) in the DS brain. The mechanisms underlying the several facets of brain development alterations in DS are largely unknown. Therefore, in the current study we exploited an in vitro model of neuronal precursor cells from the Ts65Dn mouse in order to dissect the APP-dependent molecular mechanisms underlying defective cell fate specification and neurite development.

EXPERIMENTAL PROCEDURES
Ts65Dn Mice Colony-Female Ts65Dn mice carrying a segmental trisomy of chromosome 16 (13,28) were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained on the original genetic background by mating them to C57BL/ 6JEi x C3SnHeSnJ (B6EiC3) F1 males. Animals were karyotyped using real-time quantitative PCR (qPCR) as previously described (29) and using PCR with primers spanning the translocation breakpoint of extra chromosome 17 16 (30). The animals had access to water and food ad libitum and were kept in a room with a 12:12-h dark/light cycle. Experiments were per-formed in accordance with the Italian and European Community law for the use of experimental animals and were approved by Bologna University Bioethical Committee. In this study all efforts were made to minimize animal suffering and to keep the number of animals used to a minimum.
In Vitro Differentiation and Treatments-Neurospheres were dissociated into a single cell suspension and plated onto poly-L-ornithine-coated 24-well chamber slides at a density of 3 ϫ 10 4 cells per well. Cells were cultured for 2 days in a DMEM/F-12 medium containing EGF (20 ng/ml), FGF (20 ng/ml), and 2% FBS and then transferred to a differentiation medium (EGF-and FGF-free plus 1% FBS) for 6 or 12 days. Every 2 days half of the medium was replaced with fresh differentiation medium. Treatments during in vitro NPC differentiation were performed as follows.
Viral Particles Transduction-NPCs were infected at day 1 post-plating with mouse APP shRNA and non-targeting shRNA control lentiviral particles (Santa Cruz Biotechnology), APP adenovirus particles (Vector Biolabs), and AICD lentiviral particles at different multiplicities of infection (m.o.i.). Twentyfour hours later the medium was replaced with a differentiation medium. We used the m.o.i. that gave an efficiency of transduction of around 50 -60% at 24 h after infection. Efficiency of transduction was evaluated by infecting cells with either GFP adenovirus or GFP lentivirus particles and counting GFP-positive cells. To test whether the new differentiated neurons and astrocytes were equally infected, we evaluated the percentage of GFP positive cells labeled with either ␤-tubulin III or GFAP 6 days after infection. We found a similar percentage of new neurons and astrocytes expressing the GFP marker, indicating that there was not a lineage-related selectivity of infection.
Silencing of Ptch1 Expression-A phosphorothioate Ptch1 antisense oligonucleotide (26) and a scrambled oligonucleotide, as a control, were added daily for 3 days after cell plating at the final concentration of 10 M.
Evaluation of neurite length was performed by using the image analysis system Image Pro Plus (Media Cybernetics, Silver Spring, MD). The average neurite length per cell was calculated by dividing the total neurite length by the number of cells counted in the areas.
Western Blotting-Total proteins from differentiated NCP cultures were obtained as previously described (32). For measurement of sAPP, the medium was collected after 6 days of differentiation. The collected medium was centrifuged at 4000 ϫ g for 10 min to remove the cellular debris, lyophilized, and resuspended in water plus PMSF (10 ml medium in 250 l of water). Protein concentration was estimated using the Lowry method (33). An equal amount of proteins (50 g from total protein extract and 300 g from the medium) were subjected to electrophoresis on a 4 -15% Mini-PROTEAN TGX™ Gel (Bio-Rad) and transferred to a Hybond ECL nitrocellulose membrane (Amersham Biosciences). The following primary antibodies were used: anti-Ptch1 rabbit polyclonal (1:500; Abcam), anti-Alzheimer precursor protein A4 clone 22C11 mouse monoclonal (catalog, MAB348 1:500; Millipore), anti p-STAT3 rabbit polyclonal (1:1000; Cell Signaling), anti-STAT3 rabbit polyclonal (1:1000; Cell Signaling), and anti-GAPDH rabbit polyclonal (1:5000; Sigma). Serial dilutions of total NPC protein extracts (10 -25-50 -100 g) were used to determine the linear detection range of the antibodies. Densitometric analysis of digitized images was performed with Scion Image software (Scion Corporation, Frederick, MD), and intensity for each band was normalized to the intensity of the corresponding GAPDH band.
sAPP␣ ELISA Assay-Measurement of ␣-secretase-cleaved sAPP␣ in the conditioned medium was undertaken using a sensitive and mouse/rat sAPP␣ kit (Demeditec Diagnostics). The assay was performed according to the manufacturer's protocol. Briefly, 30 l of conditioned medium samples were loaded in capture antibody (anti rodent APP; clone 597)-coated wells of the ELISA plate and incubated overnight at 4°C. After several washes the wells were incubated with detection antibody (HRPconjugated anti-rodent APP; clone 18) for 30 min at 4°C. After additional washes, chromogen solution was added to each well and kept at room temperature for 30 min. The reaction was stopped by adding 1 N H 2 SO 4 , and colorimetric signals were recorded (450 nm) using a Model 550 microplate reader (Bio-Rad). A standard curve with known amounts of recombinant rodent sAPP␣ was also generated. sAPP␣ values were normalized by the total protein content of the conditioned medium samples and plotted as pg/mg of protein present in conditioned medium samples.
Statistical Analysis-Results are presented as the means Ϯ S.E. Statistical significance was assessed by two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test or by the two-tailed Student's t test. A probability level of p Ͻ 0.05 was considered to be statistically significant.

Neuronal Precursor Cell Cultures from the Ts65Dn Mouse Exhibit Altered Cell Fate Specification and Reduced Neurite
Length, Similar to the in Vivo Condition-We previously reported that NPCs from the subventricular zone of Ts65Dn mice exhibited a defect in cell fate specification at the third passage (P3) in culture similar to that observed in vivo (26). The number of neurons generated from trisomic NPCs was notably smaller compared with that from controls, and the number of astrocytes was larger (26). To determine whether NPCs from Ts65Dn mice exhibit changes in fate acquisition related to the passage in culture, we compared the number of neurons (␤-tubulin III-positive cells), astrocytes (GFAP-positive cells), and oligodendrocytes (RIP-positive cells) produced by Ts65Dn and euploid NPCs at the first two passages (P1, P2) after 6 days of differentiation. Consistent with previous evidence (34), we observed a passage-dependent reduction in the absolute number of differentiated cells (number of ␤-tubulin III plus GFAPpositive cells: P1 Ͼ P2) (Fig. 1A). However, the number of neurons generated from trisomic NPCs was smaller compared with that generated from controls (Ϫ50%; p ϭ 7.5 ϫ 10 Ϫ6 , two-tailed t test), and the number of astrocytes was larger (ϩ55%; p ϭ 4.3 ϫ 10 Ϫ6 , two-tailed t test) at both P1 and P2 (Fig. 1A). These results suggest that trisomic NPCs have an intrinsic defect in cell fate specification that is independent from the passage in culture. At both passages, the total number of undifferentiated cells (cells that did not express markers of either cell lineage) was larger (ϩ20%; p ϭ 2.9 ϫ 10 Ϫ3 , two-tailed t test) in Ts65Dn than in control NPCs (Neither; Fig. 1A), suggesting that the trisomic condition partially inhibits differentiation of NPCs. To verify whether the reduced number of differentiated cells generated by trisomic NPCs was due to a delay in the differentiation process, we evaluated the cellular fate after a prolonged time (12 days) of differentiation. We found that the relative number of neurons and astrocytes over total cell number after 12 days was similar to that observed after 6 days in both Ts65Dn and control NPC cultures (Fig. 1A), indicating that Ts65Dn NPCs had more undifferentiated cells even after 12 days in culture. This indicates that the reduced neuronal cell fate specification in trisomic NPCs was not due to a differentiation delay. No differences were found in the percentage of apoptotic cells between Ts65Dn and control cultures (data not shown), suggesting that the reduced neuronogenesis of trisomic NPCs was not attributable to an increased death of new generated neurons. At both P1 and P2 very few cells (0.5% at P1, p ϭ 4.6 ϫ 10 Ϫ14 ; 1.2% at P2, p ϭ 6.03 ϫ 10 Ϫ7 , two-tailed t test) expressed RIP in control NPC cultures (Fig. 1B). In agreement with recent evidence in human trisomic NPCs (35), we found that trisomic NPCs from Ts65Dn mice generated more oligodendrocytes (ϩ30%; p ϭ 0.0037, two-tailed t test) compared with control NPCs (Fig. 1B).
An assessment of neurite outgrowth revealed that neurons generated from trisomic NPCs were less differentiated compared with euploid neurons. ␤-Tubulin III-positive cells showed a 55% (p ϭ 1.8 ϫ 10 Ϫ10 , two-tailed t test) reduction in average neurite length in Ts65Dn versus control neurons (Fig.  1, C and D). As expected, after 12 days of differentiation, neurite length increased in both trisomic and control neurons. Importantly, trisomic neurons retained the same percentage of reduction in neurite length compared with controls ( Fig. 1, C and D), indicating that the reduced differentiation of trisomic neurons was not due to a differentiation delay.
These results indicate that cultures of differentiated NPCs are a suitable model for studying the mechanisms underlying neurogenesis impairment in DS because they exhibit impaired acquisition of a neuronal phenotype and neuronal maturation, similar to the in vivo condition (5, 17-19, 32, 36). In view of the high number of differentiated cells at P1, we used P1 cultures for the further steps of our study.

APP/AICD-dependent Ptch1 Overexpression in Differentiated Neuronal Precursor Cell Cultures from the Ts65Dn Mouse-
We have recently demonstrated that neuronal precursors from the Ts65Dn mouse exhibited Shh pathway derangement due to overexpression of Ptch1 (26). Ptch1 overexpression was due to increased levels of AICD, a transcription-promoting fragment of the trisomic gene APP. We first sought to establish whether these defects are retained in differentiated trisomic NPC cultures. We found that trisomic cultures exhibited overexpression of APP and Ptch1, both at RNA and protein levels (Fig. 2, A  and B). To verify whether Ptch1 overexpression in trisomic cultures was APP-dependent, we reduced APP expression by using lentiviral-mediated RNA interference. At increasing m.o.i. (0.5, 2.5, and 5), APP expression in trisomic NPCs was progressively reduced (Fig. 2C). In parallel with the reduction in APP expression, there was a reduction in Ptch1 protein level (Fig. 2C). In contrast, in euploid NPCs a reduction in APP expression through RNA interference (m.o.i. 5.0) was not accompanied by a concomitant reduction in Ptch1 expression (Fig. 2D). This is in line with previous evidence showing that in euploid neurospheres AICD does not bind to the Ptch1 promoter (Trazzi et al. (26)), suggesting that AICD can modulate Ptch1 expression only when overexpressed. We next examined the effect of increased levels of APP on Ptch1 expression in euploid NPCs. After infection with recombinant adenoviruses containing the APP sequence, the increase in APP expression was paralleled by a concomitant increase in Ptch1 expression (Fig. 2D), confirming that high levels of APP cause an increase in Ptch1 expression.

Normalization of APP Expression in Neuronal Precursor Cell Cultures from the Ts65Dn Mouse Restores Cell Fate Specification and Neurite
Length-The finding that APP (and Ptch1) overexpression is retained in differentiated trisomic cells supports the hypothesis that the defective differentiation of trisomic NPCs may be APP-dependent. We found that in trisomic NPC cultures, APP interference treatments significantly increased the number of neurons and reduced the number of astrocytes compared with untreated trisomic cultures (Fig. 3, A  and C). At m.o.i. 2.5, the multiplicity of infection at which APP and Ptch1 expression in trisomic NPCs became similar to that of the euploid NPCs (Fig. 2C), cell fate specification of trisomic NPCs completely recovered (Fig. 3, A and C). APP overexpression in euploid cultures was accompanied by a reduction in the number of neurons and an increase in the number of astrocytes (Fig. 3, A and C). Taken together these data strongly suggest that APP overexpression underlies the aberrant cell fate specification that characterizes trisomic NPCs.
To assess whether the APP-dependent defective differentiation of trisomic NPCs was due to AICD accumulation, we overexpressed AICD in euploid cultures. We found that AICD overexpression significantly decreased the number of neurons compared with untreated euploid cultures (Fig. 3, A and C). In contrast, the number of astrocytes did not undergo an increase (Fig. 3, A and C), suggesting that AICD/Ptch1 signaling in trisomic NPCs is responsible for the reduced neuronogensis but does not underpin increased astrogliogenesis.
We next determined whether impaired neurite outgrowth in trisomic NPCs was also dependent on overexpression of the APP system. The addition of m.o.i. 0.5 and 2.5 APP shRNA lentiviral particles to trisomic NPC cultures induced a dose-dependent increase in neurite outgrowth (Fig. 3, B  and D). Restoration of APP expression in trisomic NPCs (m.o.i. 2.5) brought about a complete recovery of neurite outgrowth (Fig. 3, B and D). Conversely, APP as well as AICD overexpression reduced neurite outgrowth in euploid cultures (Fig. 3, B and D), which is in line with the reduced neurite length found in trisomic NPCs.

Shh Pathway Activation in Neuronal Precursor Cell Cultures from the Ts65Dn Mouse Restores Neuronogenesis and Neurite
Length but Not Astrogliogenesis-To determine whether derangement of the Shh pathway due to Ptch1 up-regulation underlies impaired cell fate specification in trisomic NPCs, we treated NPCs with SAG, an agonist for Smoothened (Smo) that relieves the inhibitory effect of Ptch1 on Smo (37) (Fig. 4C). Although increased activation of the Shh pathway by SAG had no effect on the percentage of new neurons and astrocytes in euploid NPCs (Fig. 4A), in treated trisomic NPCs the percentage of new neurons was strongly enhanced (Fig. 4A, ϩ98%; p ϭ 0.00054, t test). In contrast, the number of astrocytes did not undergo a concomitant reduction, suggesting that a defective Shh pathway in trisomic NPCs is responsible for the reduced neuronogensis but does not underpin increased astrogliogenesis (Fig. 4A). Exposure of euploid NPCs to cyclopamine, an inhibitor of Smo, reduced the number of new neurons without affecting that of astrocytes (Fig. 4A). In contrast, treatment with cyclopamine did not reduce neuronogenesis in trisomic NPCs (Fig. 4A), which suggests a full inhibition of Smo due to Ptch1  overexpression. To gain further evidence that in trisomic NPCs the Shh pathway is functionally inactive due to Ptch1 up-regulation, we reduced Ptch1 expression by using an antisense oligonucleotide that restores Ptch1 overexpression in trisomic NPCs to euploid levels (26). We found that although antisense treatment restored the number of new neurons in trisomic NPCs, with no effect on the number of astrocytes (Fig. 4A), it had no effect on the percentage of new neurons and astrocytes in euploid NPCs (Fig. 4A). In view of the role of the Shh pathway in neuronal maturation (38,39), we sought to determine whether Ptch1 up-regulation also underlies reduced neurite length of trisomic NPCs. We measured neurite length of the new neurons after either activation of the Shh pathway by SAG or inhibition by cyclopamine. Although in euploid NPC cultures treatment with SAG had no effect on neurite length (Fig.  4B), in treated trisomic NPC cultures the neurite length of the new-generated neurons underwent a large increase (Fig. 4B). Conversely, inhibition of the Shh pathway by cyclopamine reduced neurite length in euploid neurons but had no effect on trisomic neurons (Fig. 4B). Although treatment with the antisense for Ptch1 completely restored neurite length in trisomic neurons (Fig. 4B), it had no effect on neurite length in euploid neurons (Fig. 4B).
The Gli transcription factors are mediators of Shh signaling and are transcriptionally regulated by it. Several studies have demonstrated that all Gli proteins have neurogenic properties (40 -42). We found that Gli1 and Gli2 were down-regulated (Ϫ66% and Ϫ40%; p ϭ 0.00029 and p ϭ 0.034, t test, respectively) in differentiated trisomic NPC cultures (Fig. 5A), whereas no differences in Gli3 expression were found between euploid and trisomic cultures (data not shown). In trisomic NPC cultures Gli1 and Gli2 expression was increased by SAG treatment (Fig. 5A). In contrast, in control cultures SAG treatment increased Gli1 expression but had no effect on that of Gli2 (Fig. 5A). Of the three family members, Gli2 had the strongest neurogenic properties (42,43). It has recently been shown that Gli2 induces neuronogenesis but not gliogenesis in neuronal stem cells by positively regulating the expression of some neurogenic basic helix-loop-helix genes such as Mash1 (43). Looking at the expression of Mash1, we found that it was downregulated (-30%; p ϭ 0.012, t test) in trisomic NPCs and that SAG treatment restored its expression (Fig. 5B). Consistent with the absence of effects on Gli2 expression, the expression of Mash1 was unaffected by SAG treatment in control NPC cultures (Fig. 5B). Looking at the expression of the astrocytic gene GFAP, we found that, consistent with immunohistochemistry data (Fig. 1A), GFAP expression was up-regulated in trisomic NPC cultures and that SAG treatment did not influence its expression (Fig. 5B).
Taken together, these results suggest that in trisomic NPCs, Shh pathway inhibition due to Ptch1 up-regulation impairs neuronogenesis and neurite length but not astrogliogenesis. This implies that APP overexpression affects astrogliogenesis through a different pathway(s).
Increased Levels of sAPP in Neuronal Precursor Cell Cultures from the Ts65Dn Mouse Increase Astrogliogenesis-It has been shown that treatment of NPCs with either sAPP␣ or sAPP␤ increases the number of cells expressing the astrocytic marker GFAP (21,22). This suggests that increased levels of sAPP, due to the triplication of APP, may be responsible for the increased astrogliogenesis that characterizes the trisomic condition. We first examined the levels of sAPP in the culture medium of euploid and trisomic differentiated cultures. We found that sAPP levels were notably higher in the medium of trisomic cultures than in that of euploid cultures (Fig. 6, A and B). To clear the effects of increased sAPP levels, we treated trisomic NPC cultures with an antibody (22C11) that, by cross-linking APP at the N-terminal domain (i.e. the domain belonging to sAPP), prevents the interaction of sAPP with numerous cell surface receptors (44). Treatment with increasing 22C11 concentrations proportionally reduced the number of GFAP-positive cells in trisomic NPC cultures, and at high doses it normalized the number of GFAP-positive cells to euploid levels (Fig. 6C). No effect was found on the number of neurons (Fig. 6C). To confirm that the increased astrogliogenesis in trisomic NPCs was due to increased sAPP levels and not to the APP/AICD/ Shh system, we inhibited APP expression using APP shRNA treatment. We concomitantly inhibited the Shh pathway (using cyclopamine) to prevent activation of the Shh pathway due to the reduction of AICD levels. We found that treated trisomic NPC cultures underwent a decrease in the number of astrocytes with no changes in the number of neurons compared with untreated trisomic cultures (Fig. 6C). These findings demonstrate that increased levels of sAPP in trisomic NPCs specifically increase astrogliogenesis without influencing neuronogenesis.
To determine whether the reduced neurite length of trisomic neurons was due to high sAPP levels, we treated trisomic NPC cultures with either increasing 22C11 concentrations or with APP shRNA plus cyclopamine. A comparison of treated with untreated trisomic NPC cultures showed no effect on neurite length (Fig. 6D), indicating that the APP/sAPP system does not impair neurite development.
The structural property of the N-terminal domain of sAPP␣ indicates that secreted soluble APP may have a growth factorlike function by interacting with cell surface receptors (45). It has recently been shown that sAPP␣ overexpression may result in enhancement of the Notch pathway (46) as well as of the IL-6 cytokine-related pathway (47), which are crucial for the acquisition of a glial phenotype (48,49). To identify the mechanism(s) by which sAPP increases astrogliogenesis in trisomic NPCs, we examined gene expression of various molecules downstream from the Notch and IL-6 cytokine-related pathways. We found no differences between trisomic and euploid NPC cultures in the expression of Hes1 (Fig. 7A), the sAPP-dependent Notch effector involved in glial differentiation (44), suggesting the lack of involvement of the Notch pathway in increased astrogliogenesis in trisomic NPCs.  . The APP/sAPP system enhances astrogliogenesis in NPCs from Ts65Dn mice through the IL-6 cytokine-related pathway. A-C, shown is evaluation by RT-qPCR of the expression of HES1, gp130, JAK1, STAT3, and GFAP in NPC cultures from Ts65Dn (n ϭ 6) and euploid (n ϭ 6) mice. Trisomic NPC cultures were treated with anti-Alzheimer precursor protein antibody clone 22C11 (1 g/ml). D, shown is a Western blot analysis of p-STAT3 expression in cultures from Ts65Dn (TS, n ϭ 4) and euploid (EU, n ϭ 4) mice. Trisomic NPC cultures were treated with the antibody 22C11 (doses, 0.5 and 1.0 g/ml). Euploid NPCs were infected with APP adenovirus particles (m.o.i. 25). p-STAT3 levels were normalized to total S⌻〈⌻3 content. Data in A-D are given as a percentage of the euploid untreated condition. Values represent the mean Ϯ S.E. *, p Ͻ 0.05; **, p Ͻ 0.01 as compared with the euploid condition; #, p Ͻ 0.05; ##, p Ͻ 0.01 as compared with the untreated trisomic condition (Bonferroni test after ANOVA).
Members of the IL-6 family of cytokines activate the signaltransducing receptor protein, gp130, and induce phosphorylation of Janus kinases (JAK) followed by activation of signal transducers and activators of transcription (STAT1 and STAT3). Then phosphorylated STAT3 translocates into the nucleus and stimulates target gene expression by interacting with the STAT3 binding element of target genes, including GFAP (see Fig. 9). JAK1 and gp130 belong to the transcriptional targets of STAT3, and consequently, the IL-6 cytokine-related pathway is efficiently modulated by an auto-regulatory positive loop (47). We found that although STAT3 gene expression levels were similar in trisomic and control NPC cultures (Fig. 7B), gp130 and JAK1 levels were significantly up-regulated in trisomic cultures (Fig. 7C), indicating an increased IL-6 cytokinerelated pathway activation in trisomic NPCs. To determine whether the increased activation of the IL-6 cytokine-related pathway in trisomic NPCs was dependent on increased levels of sAPP, we treated trisomic NPC cultures with the 22C11 antibody to inhibit sAPP interaction with target receptors. We found that treatment with 22C11 drastically reduced the expression of gp130 and JAK1 as well as that of GFAP (Fig. 7, B and C). The phosphorylation status of STAT3 is known to be crucial for its function (50). Although we did not observe a difference in the gene expression level of STAT3, we found that in trisomic NPCs the phosphorylation status of STAT3 was increased in comparison with euploid NPCs. Treatment with the 22C11 antibody dose-dependently suppressed this increase (Fig. 7D). A similar increase in STAT3 phosphorylation was observed in euploid NPCs infected with APP (Fig. 7D). The ensemble of these data suggests that increased sAPP levels hyperactivate the IL-6 cytokine-related pathway, thereby increasing astrogliogenesis.
Inhibition of the Shh Pathway and Activation of the IL-6 Cytokine-related Pathway in the Trisomic Brain-The results obtained here in trisomic cultures suggest that two distinct pathways mediate the reduced neuronogenesis and the increased astrogliogenesis of trisomic NPCs. To confirm the results obtained in vitro, we examined the activity of these pathways in the brain of the Ts65Dn mouse. We had previously obtained evidence that in the subventricular zone and hippocampal dentate gyrus of Ts65Dn mice Ptch1 had a higher expression compared with that in euploid mice (26), suggesting derangement of the Shh pathway in these regions. Here, we analyzed the expression of the Shh-target pro-neuronal gene Mash1 in hippocampal homogenates from neonate (postnatal day 2) Ts65Dn and euploid mice. In Ts65Dn mice we found that, in parallel with the overexpression of the trisomic gene APP, there was a reduced expression of Mash1 (Fig. 8A). We additionally found an increase in the expression of JAK1 and gp130 as well as of GFAP (Fig. 8A) and in the phosphorylation status of STAT3 (Fig. 8B).

DISCUSSION
This study shows that the triplicated gene APP is involved in both neuronogenesis and astrogliogenesis alterations in trisomic NPCs. Our results suggest that the AICD negatively regulates neuronogenesis and neurite length by inhibiting the Shh pathway, whereas the sAPP fragments increase astrogliogenesis via activation of the IL-6 cytokine-related pathway. These results provide novel insight into the mechanisms contributing to brain development alterations in DS.
The APP/AICD-dependent Alteration of the Shh Pathway Impairs Neuronogenesis and Neuronal Maturation of Trisomic NPCs-We have previously reported that increased levels of AICD derange the Shh pathway through Ptch1 overexpression and, hence, inhibit proliferation in trisomic NPCs (26). We found here that Ptch1 up-regulation is also present in differentiated trisomic NPCs due to AICD overexpression and that the AICD/Ptch1 system negatively influences neuronogenesis and neurite outgrowth by inhibiting the Shh pathway. Importantly, Shh pathway activation, induced either by SAG or by the silencing of Ptch1 expression, restores neuronogenesis and neurite length but not astrogliogenesis. These results indicate that the Shh pathway selectively promotes neural progenitor cell differentiation into neurons. This is in agreement with a recent study showing that Shh activation, through carbamylated erythropoietin (CEPO), specifically enhances neuronogenesis and promotes neurite outgrowth (51).
Shh pathway activation leads to the breakdown of a large protein complex formed by Fused, Sufu, and Glis in the cytoplasm and releases the Gli transcription factors. The released Glis translocate into the nucleus, resulting in transcriptional activation of specific target genes, including the Glis themselves. Gli proteins (Gli1, Gli2, and Gli3) were recently shown to be involved during neurogenesis in vivo, creating a dynamic physical network (41). Of the three family members, Gli2 had the strongest neurogenic properties (42,52). In particular, it has been shown that overexpression of Gli2 induces neuronogenesis, but not gliogenesis in P19 EC cells, and increases the expression of the pro-neurogenic gene Mash1 (43). We found here that two of the Gli proteins (Gli1 and Gli2) were downregulated in differentiated trisomic NPCs and that treatment with an agonist of the Shh pathway (SAG) increased Gli1 and Gli2 expression. Evidence from microarray database analysis through the web-based NextBio TM software shows down-regulation of Gli2 in trisomy 21 and the Ts65Dn model, suggesting the involvement of this gene in the aberrant DS phenotype. Consistent with this hypothesis, our data suggest that downregulation of Gli2 in trisomic NPCs may underlie neuronogenesis impairment. The finding that after Shh pathway activation Gli1 expression increased in control cultures without a concomitant increase in neuronogenesis makes it unlikely that Gli1 is responsible for neuronogenesis reduction in trisomic cells.
Pro-neuronal basic helix-loop-helix transcription factors, including Mash1, promote neural progenitor cell differentiation into neurons (53). The present study shows that in trisomic NPCs, impairment of the Shh pathway was associated with down-regulation of Mash1 expression and that activation of the Shh signaling pathway restored Mash1 expression. This evidence suggests that a reduced expression of Glis and, hence, of Mash1 may underlie defective acquisition of a neuronal phenotype and impairment of neurite development in trisomic NPCs. This is in agreement with evidence showing that silencing of endogenous Mash1 suppresses Shh-promoted neuronal differentiation (51).
The APP/sAPP-dependent Alteration of the IL-6 Cytokine-related Pathway Impairs Astrogliogenesis of Trisomic NPCs-The possibility that increased astrogliogenesis is due to APP overexpression in DS is supported by recent evidence showing that human neuronal precursors overexpressing APP exhibit increased GFAP and decreased MAP2 expression (54). APP is a very complex molecule that is the source of numerous fragments with varying effects during brain development. In nonpathological situations, APP is predominantly cleaved by the ␣-secretase within the ␤-amyloid sequence to release a soluble form of APP, sAPP. sAPP is normally present in brain tissues and circulates in the cerebrospinal fluid (55). A number of in vitro studies have attempted to shed light on the physiological functions of sAPP in the brain, showing that sAPP enhances synaptogenesis, neurite outgrowth, cell survival, adhesion, and proliferation of embryonic neural stem cells (56,57). In addition, recent in vitro and in vivo evidence shows that sAPP administration promotes astrogliogenesis (21,22,47). Our findings suggest that high levels of sAPP or cell surface-associated APP have a role in the increased astrocytic differentiation that characterizes trisomic NPCs. We found no effect of the APP/sAPP system on neuronogenesis or neurite length in trisomic NPCs, suggesting a specific role of sAPP as a gliogenic factor.
The astrocyte differentiation process of NPCs appears to be tightly modulated by various extrinsic (e.g. leukemia inhibitory factor, ciliary neurotrophic factor, Notch, and bone morphogenetic proteins) and intrinsic (e.g. Ngn1, Ngn2, and DNA methylation) factors. A recent study has revealed that the IL-6/gp130 signaling pathway plays a pivotal role in astrocyte differentiation of NPCs (47). Based on this evidence, it has been postulated that sAPP may affect the cell fate of neural precursor cells in DS via the activation of the IL-6/gp130 signaling pathway (47). Our data confirm this suggestion, showing that increased levels of APP/sAPP in trisomic NPCs enhance the acquisition of an astrocytic phenotype via the activation of the IL-6/gp130 signaling pathway. Cross-talk of the Notch and JAK/STAT pathways in the induction of glial differentiation in NPCs through physical interaction between Hes1 and JAK2 has recently been reported (44,58). It has been reported that treatment with exogenous sAPP induces glial differentiation of the human embryonal carcinoma cell line NT-2/D1 via the Notch signaling pathway (44). However, we did not find an sAPP-dependent Hes1 up-regulation in trisomic NPCs, suggesting that activation of the Notch signaling cascade is not involved in sAPPinduced glial differentiation in trisomic NPCs. This discrepancy may be due either to the different cellular system used, primary NPCs versus the NT-2/D1 cell line (44), and/or to different levels of sAPP accumulated in trisomic cultures in comparison with that added to the culture medium (44).
When ligands such as leukemia inhibitory factor and ciliary neurotrophic factor bind to the IL-6 cytokine-related pathway receptors, subsequent phosphorylation of gp130, JAK, and STAT3 occurs. Then, phosphorylated STAT3 translocates into the nucleus and stimulates target gene expression by interacting with the STAT3 binding element of the target genes, such as GFAP. We found that the enhanced expression of gp130, JAK1, and GFAP in trisomic NPCs was restored by treatment with an antibody that recognizes the N-terminal region of APP (sAPP). This suggests that trisomy-dependent high levels of sAPP may exert their effects through the IL-6 cytokine-related pathway. In line with data obtained in trisomic NPC cultures, we found up-regulation of gp130 and JAK1 in vivo in neuronal precursors of Ts65Dn mice. Taken together the current results strongly suggest that in trisomic NPCs high levels of sAPP may activate the gp130/JAK/STAT signaling, and as a result, STAT3 may promote transcription of GFAP.
Conclusions-The formation of the mammalian nervous system takes place via a number of developmental steps, including cell proliferation, cell-fate decision, and postmitotic neuronal maturation. All these processes are compromised in the DS brain. The mechanisms underlying these defects are presently poorly understood. Previous work (26) and current results suggest that APP triplication plays a role in the alteration of brain development in the Ts65Dn mouse model of DS.
An important issue deals with the contribution of other triplicated genes in brain development and intellectual disability in DS. According to recent evidence, the Ts1Cje mouse model, which has a shorter chromosomal triplication that does not include APP, shows reduced proliferation and differentiation of neuronal precursor cells from the subventricular zone (59) and behavioral impairment (60), similar to the Ts65Dn mouse.
However, the Ts1Cje mouse shows spine and connectivity alterations (60) and reduction in cerebellar granule cell number (61) less severe than the Ts65Dn mouse. Interestingly a high resolution analysis of human segmental trisomies suggests that more than one mental retardation critical region of HSA21 exists (62). Thus, many different triplicated genes are likely to contribute to the brain and behavioral phenotype in DS. Triplication of different genes may culminate into similar effects by acting on multiple, intersecting/converging pathways. This may explain, for instance, why APP triplication affects but is not essential for some of the trisomic phenotypes. The development of a new mouse strain obtained by crossing APP transgenic with Ts1Cje mice may provide additional clues as to the role of APP on brain phenotype in the trisomic condition.
Based on our present and previous findings we propose a model for the APP-mediated developmental defects in the Ts65Dn mouse (Fig. 9). These findings indicate that a common molecular denominator may be involved in the impairment of cell proliferation, cell fate specification, and neuronal maturation. These results may help to understand the complex pathophysiological mechanism of DS and give an indication to possible new therapeutic strategies.