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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*

Open AccessPublished:July 19, 2013DOI:https://doi.org/10.1074/jbc.M113.451088
      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.
      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.

      Introduction

      Down syndrome (DS)
      The abbreviations used are: DS
      Down syndrome
      APP
      amyloid precursor protein
      sAPP
      secreted APP
      AICD
      APP intracellular domain
      Shh
      Sonic Hedgehog
      qPCR
      quantitative PCR
      NPC
      neuronal precursors
      m.o.i.
      multiplicity of infection
      SAG
      benzo[b]thiophene-2-carboxamide,3-chloro-N-[4-(methylamino)cyclohexyl]-N-{[3-(4 pyridinyl)phenyl]methyl}-(9CI)
      GFAP
      glial fibrillary acidic protein
      ANOVA
      analysis of variance
      gp130
      glycoprotein 130
      Ptch1
      patched1
      Smo
      Smoothened.
      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 (
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      ,
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      Mental retardation and associated neurological dysfunctions in Down syndrome. A consequence of dysregulation in critical chromosome 21 genes and associated molecular pathways.
      ). 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 (
      • Ross M.H.
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      Down's syndrome. Is there a decreased population of neurons?.
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      Brain growth in Down syndrome subjects 15–22 weeks of gestational age and birth to 60 months.
      ). 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 (
      • Contestabile A.
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      Cell cycle alteration and decreased cell proliferation in the hippocampal dentate gyrus and in the neocortical germinal matrix of fetuses with Down syndrome and in Ts65Dn mice.
      ,
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      • Santini D.
      • Gualtieri F.
      • Ciani E.
      • Bartesaghi R.
      Neurogenesis impairment and increased cell death reduce total neuron number in the hippocampal region of fetuses with Down syndrome.
      ,
      • Guidi S.
      • Ciani E.
      • Bonasoni P.
      • Santini D.
      • Bartesaghi R.
      Widespread proliferation impairment and hypocellularity in the cerebellum of fetuses with Down syndrome.
      ). This proliferation impairment is worsened by impaired cell fate specification with a reduction in neuronogenesis and an increase in astrogliogenesis (
      • Contestabile A.
      • Fila T.
      • Ceccarelli C.
      • Bonasoni P.
      • Bonapace L.
      • Santini D.
      • Bartesaghi R.
      • Ciani E.
      Cell cycle alteration and decreased cell proliferation in the hippocampal dentate gyrus and in the neocortical germinal matrix of fetuses with Down syndrome and in Ts65Dn mice.
      ,
      • Guidi S.
      • Bonasoni P.
      • Ceccarelli C.
      • Santini D.
      • Gualtieri F.
      • Ciani E.
      • Bartesaghi R.
      Neurogenesis impairment and increased cell death reduce total neuron number in the hippocampal region of fetuses with Down syndrome.
      ,
      • Guidi S.
      • Ciani E.
      • Bonasoni P.
      • Santini D.
      • Bartesaghi R.
      Widespread proliferation impairment and hypocellularity in the cerebellum of fetuses with Down syndrome.
      ). 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 (
      • Becker L.E.
      Synaptic dysgenesis.
      ,
      • Takashima S.
      • Ieshima A.
      • Nakamura H.
      • Becker L.E.
      Dendrites, dementia, and the Down syndrome.
      ,
      • Takashima S.
      • Becker L.E.
      • Armstrong D.L.
      • Chan F.
      Abnormal neuronal development in the visual cortex of the human fetus and infant with Down's syndrome. A quantitative and qualitative Golgi study.
      ,
      • Schulz E.
      • Scholz B.
      Neurohistological findings in the parietal cortex of children with chromosome aberrations.
      ,
      • Prinz M.
      • Prinz B.
      • Schulz E.
      The growth of non-pyramidal neurons in the primary motor cortex of man. A Golgi study.
      ). 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 (
      • Reeves R.H.
      • Irving N.G.
      • Moran T.H.
      • Wohn A.
      • Kitt C.
      • Sisodia S.S.
      • Schmidt C.
      • Bronson R.T.
      • Davisson M.T.
      A mouse model for Down syndrome exhibits learning and behaviour deficits.
      ). Similar to individuals with DS, the Ts65Dn mouse shows severe neurogenesis impairment in numerous brain regions (
      • Contestabile A.
      • Fila T.
      • Ceccarelli C.
      • Bonasoni P.
      • Bonapace L.
      • Santini D.
      • Bartesaghi R.
      • Ciani E.
      Cell cycle alteration and decreased cell proliferation in the hippocampal dentate gyrus and in the neocortical germinal matrix of fetuses with Down syndrome and in Ts65Dn mice.
      ,
      • Haydar T.F.
      • Nowakowski R.S.
      • Yarowsky P.J.
      • Krueger B.K.
      Role of founder cell deficit and delayed neuronogenesis in microencephaly of the trisomy 16 mouse.
      ,
      • Lorenzi H.A.
      • Reeves R.H.
      Hippocampal hypocellularity in the Ts65Dn mouse originates early in development.
      ,
      • Roper R.J.
      • Baxter L.L.
      • Saran N.G.
      • Klinedinst D.K.
      • Beachy P.A.
      • Reeves R.H.
      Defective cerebellar response to mitogenic Hedgehog signaling in Down syndrome mice.
      ,
      • Chakrabarti L.
      • Galdzicki Z.
      • Haydar T.F.
      Defects in embryonic neurogenesis and initial synapse formation in the forebrain of the Ts65Dn mouse model of Down syndrome.
      ) and less branched and less spinous dendrites (
      • Benavides-Piccione R.
      • Ballesteros-Yáñez I.
      • de Lagrán M.M.
      • Elston G.
      • Estivill X.
      • Fillat C.
      • Defelipe J.
      • Dierssen M.
      On dendrites in Down syndrome and DS murine models. A spiny way to learn.
      ,
      • Belichenko P.V.
      • Masliah E.
      • Kleschevnikov A.M.
      • Villar A.J.
      • Epstein C.J.
      • Salehi A.
      • Mobley W.C.
      Synaptic structural abnormalities in the Ts65Dn mouse model of Down Syndrome.
      ).
      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 (
      • Zhou Z.D.
      • Chan C.H.
      • Ma Q.H.
      • Xu X.H.
      • Xiao Z.C.
      • Tan E.K.
      The roles of amyloid precursor protein (APP) in neurogenesis. Implications to pathogenesis and therapy of Alzheimer disease.
      ), 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 (
      • Zhou Z.D.
      • Chan C.H.
      • Ma Q.H.
      • Xu X.H.
      • Xiao Z.C.
      • Tan E.K.
      The roles of amyloid precursor protein (APP) in neurogenesis. Implications to pathogenesis and therapy of Alzheimer disease.
      ,
      • Baratchi S.
      • Evans J.
      • Tate W.P.
      • Abraham W.C.
      • Connor B.
      Secreted amyloid precursor proteins promote proliferation and glial differentiation of adult hippocampal neural progenitor cells.
      ,
      • Kwak Y.D.
      • Brannen C.L.
      • Qu T.
      • Kim H.M.
      • Dong X.
      • Soba P.
      • Majumdar A.
      • Kaplan A.
      • Beyreuther K.
      • Sugaya K.
      Amyloid precursor protein regulates differentiation of human neural stem cells.
      ), whereas AICD negatively modulates proliferation and maturation of neural precursors (
      • Zhou Z.D.
      • Chan C.H.
      • Ma Q.H.
      • Xu X.H.
      • Xiao Z.C.
      • Tan E.K.
      The roles of amyloid precursor protein (APP) in neurogenesis. Implications to pathogenesis and therapy of Alzheimer disease.
      ). APP is cleaved by three types of proteases, which are designated α-, β-, and γ-secretases and which may result in functionally distinct outcomes. The non-amyloidogenic APP processing pathway involves proteolytic cleavages exerted by α- and γ-secretases resulting in the generation of N-terminal sAPPα and C-terminal fragments including P3, CTF83, and the intracellular domain (AICD). The alternative amyloidogenic APP processing pathway involves proteolytic cleavages exerted by β- and γ-secretases resulting in the generation of sAPPβ and C-terminal fragments including Aβ, CTF99, and AICD.
      AICD has been shown to be involved in the transcriptional regulation of various genes (
      • Cao X.
      • Südhof T.C.
      A transcriptionally active complex of APP with Fe65 and histone acetyltransferase Tip60.
      ,
      • Jhoo J.H.
      • Kim H.C.
      • Nabeshima T.
      • Yamada K.
      • Shin E.J.
      • Jhoo W.K.
      • Kim W.
      • Kang K.S.
      • Jo S.A.
      • Woo J.I.
      β-Amyloid (1–42)-induced learning and memory deficits in mice. Involvement of oxidative burdens in the hippocampus and cerebral cortex.
      ), including patched1 (Ptch1) (
      • Müller T.
      • Meyer H.E.
      • Egensperger R.
      • Marcus K.
      The amyloid precursor protein intracellular domain (AICD) as modulator of gene expression, apoptosis, and cytoskeletal dynamics relevance for Alzheimer's disease.
      ). 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 (
      • Trazzi S.
      • Mitrugno V.M.
      • Valli E.
      • Fuchs C.
      • Rizzi S.
      • Guidi S.
      • Perini G.
      • Bartesaghi R.
      • Ciani E.
      APP-dependent up-regulation of Ptch1 underlies proliferation impairment of neural precursors in Down syndrome.
      ). We found that Ptch1 overexpression was correlated to increased levels of AICD (
      • Trazzi S.
      • Mitrugno V.M.
      • Valli E.
      • Fuchs C.
      • Rizzi S.
      • Guidi S.
      • Perini G.
      • Bartesaghi R.
      • Ciani E.
      APP-dependent up-regulation of Ptch1 underlies proliferation impairment of neural precursors in Down syndrome.
      ), 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 (
      • Sánchez-Camacho C.
      • Bovolenta P.
      Emerging mechanisms in morphogen-mediated axon guidance.
      ), 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 (
      • Zhou Z.D.
      • Chan C.H.
      • Ma Q.H.
      • Xu X.H.
      • Xiao Z.C.
      • Tan E.K.
      The roles of amyloid precursor protein (APP) in neurogenesis. Implications to pathogenesis and therapy of Alzheimer disease.
      ) 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.

      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 (
      • Trazzi S.
      • Mitrugno V.M.
      • Valli E.
      • Fuchs C.
      • Rizzi S.
      • Guidi S.
      • Perini G.
      • Bartesaghi R.
      • Ciani E.
      APP-dependent up-regulation of Ptch1 underlies proliferation impairment of neural precursors in Down syndrome.
      ). 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 (
      • Wang L.
      • Zhang Z.G.
      • Gregg S.R.
      • Zhang R.L.
      • Jiao Z.
      • LeTourneau Y.
      • Liu X.
      • Feng Y.
      • Gerwien J.
      • Torup L.
      • Leist M.
      • Noguchi C.T.
      • Chen Z.Y.
      • Chopp M.
      The Sonic hedgehog pathway mediates carbamylated erythropoietin-enhanced proliferation and differentiation of adult neural progenitor cells.
      ).
      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 (
      • Nguyen V.
      • Chokas A.L.
      • Stecca B.
      • Ruiz i Altaba A.
      Cooperative requirement of the Gli proteins in neurogenesis.
      ). Of the three family members, Gli2 had the strongest neurogenic properties (
      • Brewster R.
      • Lee J.
      • Ruiz i Altaba A.
      Gli/Zic factors pattern the neural plate by defining domains of cell differentiation.
      ,
      • Ruiz i Altaba A.
      Combinatorial Gli gene function in floor plate and neuronal inductions by Sonic hedgehog.
      ). 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 (
      • Voronova A.
      • Fischer A.
      • Ryan T.
      • Al Madhoun A.
      • Skerjanc I.S.
      Ascl1/Mash1 is a novel target of Gli2 during Gli2-induced neurogenesis in P19 EC cells.
      ). We found here that two of the Gli proteins (Gli1 and Gli2) were down-regulated 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 NextBioTM 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 down-regulation 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 (
      • Ma Q.
      • Sommer L.
      • Cserjesi P.
      • Anderson D.J.
      Mash1 and neurogenin1 expression patterns define complementary domains of neuroepithelium in the developing CNS and are correlated with regions expressing notch ligands.
      ). 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 (
      • Wang L.
      • Zhang Z.G.
      • Gregg S.R.
      • Zhang R.L.
      • Jiao Z.
      • LeTourneau Y.
      • Liu X.
      • Feng Y.
      • Gerwien J.
      • Torup L.
      • Leist M.
      • Noguchi C.T.
      • Chen Z.Y.
      • Chopp M.
      The Sonic hedgehog pathway mediates carbamylated erythropoietin-enhanced proliferation and differentiation of adult neural progenitor cells.
      ).

      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 (
      • Lu J.
      • Esposito G.
      • Scuderi C.
      • Steardo L.
      • Delli-Bovi L.C.
      • Hecht J.L.
      • Dickinson B.C.
      • Chang C.J.
      • Mori T.
      • Sheen V.
      S100B and APP promote a gliocentric shift and impaired neurogenesis in Down syndrome neural progenitors.
      ). APP is a very complex molecule that is the source of numerous fragments with varying effects during brain development. In non-pathological situations, APP is predominantly cleaved by the α-secretase within the β-amyloid sequence to release a soluble form of APP, sAPP. sAPP is normally present in brain tissues and circulates in the cerebrospinal fluid (
      • Palmert M.R.
      • Podlisny M.B.
      • Witker D.S.
      • Oltersdorf T.
      • Younkin L.H.
      • Selkoe D.J.
      • Younkin S.G.
      The β-amyloid protein precursor of Alzheimer disease has soluble derivatives found in human brain and cerebrospinal fluid.
      ). 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 (
      • Mattson M.P.
      Cellular actions of β-amyloid precursor protein and its soluble and fibrillogenic derivatives.
      ,
      • Ohsawa I.
      • Takamura C.
      • Morimoto T.
      • Ishiguro M.
      • Kohsaka S.
      Amino-terminal region of secreted form of amyloid precursor protein stimulates proliferation of neural stem cells.
      ). In addition, recent in vitro and in vivo evidence shows that sAPP administration promotes astrogliogenesis (
      • Baratchi S.
      • Evans J.
      • Tate W.P.
      • Abraham W.C.
      • Connor B.
      Secreted amyloid precursor proteins promote proliferation and glial differentiation of adult hippocampal neural progenitor cells.
      ,
      • Kwak Y.D.
      • Brannen C.L.
      • Qu T.
      • Kim H.M.
      • Dong X.
      • Soba P.
      • Majumdar A.
      • Kaplan A.
      • Beyreuther K.
      • Sugaya K.
      Amyloid precursor protein regulates differentiation of human neural stem cells.
      ,
      • Kwak Y.D.
      • Dantuma E.
      • Merchant S.
      • Bushnev S.
      • Sugaya K.
      Amyloid-β precursor protein induces glial differentiation of neural progenitor cells by activation of the IL-6/gp130 signaling pathway.
      ). 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 (
      • Kwak Y.D.
      • Dantuma E.
      • Merchant S.
      • Bushnev S.
      • Sugaya K.
      Amyloid-β precursor protein induces glial differentiation of neural progenitor cells by activation of the IL-6/gp130 signaling pathway.
      ). 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 (
      • Kwak Y.D.
      • Dantuma E.
      • Merchant S.
      • Bushnev S.
      • Sugaya K.
      Amyloid-β precursor protein induces glial differentiation of neural progenitor cells by activation of the IL-6/gp130 signaling pathway.
      ). 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 (
      • Kwak Y.D.
      • Marutle A.
      • Dantuma E.
      • Merchant S.
      • Bushnev S.
      • Sugaya K.
      Involvement of notch signaling pathway in amyloid precursor protein induced glial differentiation.
      ,
      • Kamakura S.
      • Oishi K.
      • Yoshimatsu T.
      • Nakafuku M.
      • Masuyama N.
      • Gotoh Y.
      Hes binding to STAT3 mediates crosstalk between Notch and JAK-STAT signalling.
      ). 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 (
      • Kwak Y.D.
      • Marutle A.
      • Dantuma E.
      • Merchant S.
      • Bushnev S.
      • Sugaya K.
      Involvement of notch signaling pathway in amyloid precursor protein induced glial differentiation.
      ). 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 sAPP-induced 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 (
      • Kwak Y.D.
      • Marutle A.
      • Dantuma E.
      • Merchant S.
      • Bushnev S.
      • Sugaya K.
      Involvement of notch signaling pathway in amyloid precursor protein induced glial differentiation.
      ), and/or to different levels of sAPP accumulated in trisomic cultures in comparison with that added to the culture medium (
      • Kwak Y.D.
      • Marutle A.
      • Dantuma E.
      • Merchant S.
      • Bushnev S.
      • Sugaya K.
      Involvement of notch signaling pathway in amyloid precursor protein induced glial differentiation.
      ).
      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 (
      • Trazzi S.
      • Mitrugno V.M.
      • Valli E.
      • Fuchs C.
      • Rizzi S.
      • Guidi S.
      • Perini G.
      • Bartesaghi R.
      • Ciani E.
      APP-dependent up-regulation of Ptch1 underlies proliferation impairment of neural precursors in Down syndrome.
      ) 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 (
      • Hewitt C.A.
      • Ling K.H.
      • Merson T.D.
      • Simpson K.M.
      • Ritchie M.E.
      • King S.L.
      • Pritchard M.A.
      • Smyth G.K.
      • Thomas T.
      • Scott H.S.
      • Voss A.K.
      Gene network disruptions and neurogenesis defects in the adult Ts1Cje mouse model of Down syndrome.
      ) and behavioral impairment (
      • Belichenko P.V.
      • Kleschevnikov A.M.
      • Salehi A.
      • Epstein C.J.
      • Mobley W.C.
      Synaptic and cognitive abnormalities in mouse models of Down syndrome. Exploring genotype-phenotype relationships.
      ), similar to the Ts65Dn mouse. However, the Ts1Cje mouse shows spine and connectivity alterations (
      • Belichenko P.V.
      • Kleschevnikov A.M.
      • Salehi A.
      • Epstein C.J.
      • Mobley W.C.
      Synaptic and cognitive abnormalities in mouse models of Down syndrome. Exploring genotype-phenotype relationships.
      ) and reduction in cerebellar granule cell number (
      • Olson L.E.
      • Roper R.J.
      • Baxter L.L.
      • Carlson E.J.
      • Epstein C.J.
      • Reeves R.H.
      Down syndrome mouse models Ts65Dn, Ts1Cje, and Ms1Cje/Ts65Dn exhibit variable severity of cerebellar phenotypes.
      ) 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 (
      • Korbel J.O.
      • Tirosh-Wagner T.
      • Urban A.E.
      • Chen X.N.
      • Kasowski M.
      • Dai L.
      • Grubert F.
      • Erdman C.
      • Gao M.C.
      • Lange K.
      • Sobel E.M.
      • Barlow G.M.
      • Aylsworth A.S.
      • Carpenter N.J.
      • Clark R.D.
      • Cohen M.Y.
      • Doran E.
      • Falik-Zaccai T.
      • Lewin S.O.
      • Lott I.T.
      • McGillivray B.C.
      • Moeschler J.B.
      • Pettenati M.J.
      • Pueschel S.M.
      • Rao K.W.
      • Shaffer L.G.
      • Shohat M.
      • Van Riper A.J.
      • Warburton D.
      • Weissman S.
      • Gerstein M.B.
      • Snyder M.
      • Korenberg J.R.
      The genetic architecture of Down syndrome phenotypes revealed by high resolution analysis of human segmental trisomies.
      ). 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.

      Acknowledgments

      We are grateful to Dr. D. Mukhopadhyay for kindly supplying the AICD plasmid.

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