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• W1993011380 abstract "The amyloid precursor protein (APP) is well studied for its role in Alzheimer disease. However, little is known about its normal function. In this study, we examined the role of APP in neural stem/progenitor cell (NSPC) proliferation. NSPCs derived from APP-overexpressing Tg2576 transgenic mice proliferated more rapidly than NSPCs from the corresponding background strain (C57Bl/6xSJL) wild-type mice. In contrast, NSPCs from APP knock-out (APP-KO) mice had reduced proliferation rates when compared with NSPCs from the corresponding background strain (C57Bl/6). A secreted factor, identified as cystatin C, was found to be responsible for this effect. Levels of cystatin C were higher in the Tg2576 conditioned medium and lower in the APP-KO conditioned medium. Furthermore, immunodepletion of cystatin C from the conditioned medium completely removed the ability of the conditioned medium to increase NSPC proliferation. The results demonstrate that APP expression stimulates NSPC proliferation and that this effect is mediated via an increase in cystatin C secretion.Background: The role of the amyloid precursor protein (APP) in neural stem/progenitor cell (NSPC) proliferation is poorly understood.Results: Immunodepletion of cystatin C from NSPC conditioned medium abrogated an effect of APP on NSPC proliferation.Conclusion: Cystatin C mediates APP-induced NSPC proliferation.Significance: The results increase understanding of mechanisms promoting NSPC survival and differentiation. The amyloid precursor protein (APP) is well studied for its role in Alzheimer disease. However, little is known about its normal function. In this study, we examined the role of APP in neural stem/progenitor cell (NSPC) proliferation. NSPCs derived from APP-overexpressing Tg2576 transgenic mice proliferated more rapidly than NSPCs from the corresponding background strain (C57Bl/6xSJL) wild-type mice. In contrast, NSPCs from APP knock-out (APP-KO) mice had reduced proliferation rates when compared with NSPCs from the corresponding background strain (C57Bl/6). A secreted factor, identified as cystatin C, was found to be responsible for this effect. Levels of cystatin C were higher in the Tg2576 conditioned medium and lower in the APP-KO conditioned medium. Furthermore, immunodepletion of cystatin C from the conditioned medium completely removed the ability of the conditioned medium to increase NSPC proliferation. The results demonstrate that APP expression stimulates NSPC proliferation and that this effect is mediated via an increase in cystatin C secretion. Background: The role of the amyloid precursor protein (APP) in neural stem/progenitor cell (NSPC) proliferation is poorly understood. Results: Immunodepletion of cystatin C from NSPC conditioned medium abrogated an effect of APP on NSPC proliferation. Conclusion: Cystatin C mediates APP-induced NSPC proliferation. Significance: The results increase understanding of mechanisms promoting NSPC survival and differentiation. Neural stem cells are self-renewing, multipotent cells that can produce all of the major cellular phenotypes in the nervous system (1Reynolds B.A. Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.Science. 1992; 255: 1707-1710Crossref PubMed Scopus (4545) Google Scholar, 2Richards L.J. Kilpatrick T.J. Bartlett P.F. De novo generation of neuronal cells from the adult mouse brain.Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 8591-8595Crossref PubMed Scopus (461) Google Scholar). Neural stem cells are important, not only because they produce the entire complement of neuronal and glial cells of the mature nervous system and continue to generate new neurons throughout life, but also because they may be useful for the therapeutic replacement of cells in neurodegenerative diseases. The mechanisms that promote neural stem or progenitor cell (NSPC) 4The abbreviations used are:NSPC, neural stem/progenitor cell; APP, β-amyloid precursor protein; sAPP, soluble APP; APP-KO, APP knock-out; Aβ, β-amyloid protein; AICD, APP intracellular domain; bFGF, basic fibroblast growth factor; AD, Alzheimer disease; EdU, 5-ethynyl-2′-deoxyuridine; ANOVA, analysis of variance. proliferation are only partially understood. Both epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) are well studied for their roles in stimulating NSPC proliferation in vitro (2Richards L.J. Kilpatrick T.J. Bartlett P.F. De novo generation of neuronal cells from the adult mouse brain.Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 8591-8595Crossref PubMed Scopus (461) Google Scholar, 3Moyse E. Segura S. Liard O. Mahaut S. Mechawar N. Microenvironmental determinants of adult neural stem cell proliferation and lineage commitment in the healthy and injured central nervous system.Curr. Stem Cell Res. Ther. 2008; 3: 163-184Crossref PubMed Scopus (46) Google Scholar). However, other autocrine growth factors produced by the NSPCs themselves may also be necessary for optimum growth. The β-amyloid precursor protein (APP) is a 110–130-kDa integral type I transmembrane glycoprotein that has been extensively studied for its role in Alzheimer disease (AD) (4Small D.H. Mok S.S. Bornstein J.C. Alzheimer's disease and Aβ toxicity: from top to bottom.Nat. Rev. Neurosci. 2001; 2: 595-598Crossref PubMed Scopus (354) Google Scholar). Despite the very large number of published studies on APP, the normal function of APP has remained a mystery. APP is encoded by a single gene located on chromosome 21 (5Patterson D. Gardiner K. Kao F.T. Tanzi R. Watkins P. Gusella J.F. Mapping of the gene encoding the β-amyloid precursor protein and its relationship to the Down syndrome region of chromosome 21.Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 8266-8270Crossref PubMed Scopus (53) Google Scholar). APP is post-translationally glycosylated and phosphorylated and can be cleaved by two major proteolytic pathways. In one pathway, sequential cleavage of APP by α- and γ-secretase generates a large ectodomain fragment (sAPPα), which is secreted into the extracellular milieu, and a small C-terminal fragment (the APP intracellular domain or AICD), which may have a role in regulating gene expression. In the other pathway, cleavage of APP by β- and γ-secretase generates a different ectodomain fragment (sAPPβ), as well as the AICD peptide. Cleavage of APP via this second pathway also generates the β-amyloid protein (Aβ) of AD (6Nunan J. Small D.H. Regulation of APP cleavage by α-, β-, and γ-secretases.FEBS Lett. 2000; 483: 6-10Crossref PubMed Scopus (419) Google Scholar). Although the normal function of APP is poorly understood, the pattern of expression of APP suggests that it may be important for neuronal growth and differentiation, not only in the developing brain but also in the mature or aging nervous system. The expression of APP has been shown to increase as the nervous system matures (7Small D.H. Nurcombe V. Moir R. Michaelson S. Monard D. Beyreuther K. Masters C.L. Association and release of the amyloid protein precursor of Alzheimer's disease from chick brain extracellular matrix.J. Neurosci. 1992; 12: 4143-4150Crossref PubMed Google Scholar). APP expression increases as NSPCs mature into neurons, and soluble APP has been reported to promote neural differentiation (8Lee J.A. Cole G.J. Generation of transgenic zebrafish expressing green fluorescent protein under control of zebrafish amyloid precursor protein gene regulatory elements.Zebrafish. 2007; 4: 277-286Crossref PubMed Scopus (33) Google Scholar, 9Freude K.K. Penjwini M. Davis J.L. LaFerla F.M. Blurton-Jones M. Soluble amyloid precursor protein induces rapid neural differentiation of human embryonic stem cells.J. Biol. Chem. 2011; 286: 24264-24274Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). APP may also play a role in later stages of neuronal development. For example, soluble APP is reported to stimulate neurite outgrowth in a variety of cell systems (10Milward E.A. Papadopoulos R. Fuller S.J. Moir R.D. Small D. Beyreuther K. Masters C.L. The amyloid protein precursor of Alzheimer's disease is a mediator of the effects of nerve growth factor on neurite outgrowth.Neuron. 1992; 9: 129-137Abstract Full Text PDF PubMed Scopus (402) Google Scholar, 11Mattson M.P. Secreted forms of β-amyloid precursor protein modulate dendrite outgrowth and calcium responses to glutamate in cultured embryonic hippocampal neurons.J. Neurobiol. 1994; 25: 439-450Crossref PubMed Scopus (142) Google Scholar, 12Small D.H. Nurcombe V. Reed G. Clarris H. Moir R. Beyreuther K. Masters C.L. A heparin-binding domain in the amyloid protein precursor of Alzheimer's disease is involved in the regulation of neurite outgrowth.J. Neurosci. 1994; 14: 2117-2127Crossref PubMed Google Scholar, 13Gakhar-Koppole N. Hundeshagen P. Mandl C. Weyer S.W. Allinquant B. Müller U. Ciccolini F. Activity requires soluble amyloid precursor protein α to promote neurite outgrowth in neural stem cell-derived neurons via activation of the MAPK pathway.Eur. J. Neurosci. 2008; 28: 871-882Crossref PubMed Scopus (86) Google Scholar, 14Chasseigneaux S. Dinc L. Rose C. Chabret C. Coulpier F. Topilko P. Mauger G. Allinquant B. Secreted amyloid precursor protein β and secreted amyloid precursor protein α induce axon outgrowth in vitro through Egr1 signaling pathway.PLoS One. 2011; 6: e16301Crossref PubMed Scopus (69) Google Scholar). Our studies have shown that APP expression is increased in the olfactory neuroepithelium at the developmental stage when neurogenesis and neurite outgrowth begin (15Clarris H.J. Key B. Beyreuther K. Masters C.L. Small D.H. Expression of the amyloid protein precursor of Alzheimer's disease in the developing rat olfactory system.Brain Res. Dev. Brain Res. 1995; 88: 87-95Crossref PubMed Scopus (50) Google Scholar). Similarly, APP has been reported to regulate a number of developmental functions including neuronal migration (16Young-Pearse T.L. Bai J. Chang R. Zheng J.B. LoTurco J.J. Selkoe D.J. A critical function for β-amyloid precursor protein in neuronal migration revealed by in utero RNA interference.J. Neurosci. 2007; 27: 14459-14469Crossref PubMed Scopus (273) Google Scholar) and cell growth (17Hornsten A. Lieberthal J. Fadia S. Malins R. Ha L. Xu X. Daigle I. Markowitz M. O'Connor G. Plasterk R. Li C. APL-1, a Caenorhabditis elegans protein related to the human β-amyloid precursor protein, is essential for viability.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1971-1976Crossref PubMed Scopus (113) Google Scholar, 18Joshi P. Liang J.O. DiMonte K. Sullivan J. Pimplikar S.W. Amyloid precursor protein is required for convergent-extension movements during Zebrafish development.Dev. Biol. 2009; 335: 1-11Crossref PubMed Scopus (60) Google Scholar). A role for APP in cell growth is supported by the rapid up-regulation of APP that occurs in response to axonal injury (19Gentleman S.M. Graham D.I. Roberts G.W. Molecular pathology of head trauma: altered β APP metabolism and the aetiology of Alzheimer's disease.Prog. Brain Res. 1993; 96: 237-246Crossref PubMed Scopus (86) Google Scholar, 20Blumbergs P.C. Scott G. Manavis J. Wainwright H. Simpson D.A. McLean A.J. Topography of axonal injury as defined by amyloid precursor protein and the sector scoring method in mild and severe closed head injury.J. Neurotrauma. 1995; 12: 565-572Crossref PubMed Scopus (284) Google Scholar, 21Itoh T. Satou T. Nishida S. Tsubaki M. Hashimoto S. Ito H. Expression of amyloid precursor protein after rat traumatic brain injury.Neurol Res. 2009; 31: 103-109Crossref PubMed Scopus (59) Google Scholar). Dystrophic neurites found around amyloid plaques are highly immunoreactive for APP (22Cras P. Kawai M. Lowery D. Gonzalez-DeWhitt P. Greenberg B. Perry G. Senile plaque neurites in Alzheimer disease accumulate amyloid precursor protein.Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 7552-7556Crossref PubMed Scopus (217) Google Scholar, 23Cochran E. Bacci B. Chen Y. Patton A. Gambetti P. Autilio-Gambetti L. Amyloid precursor protein and ubiquitin immunoreactivity in dystrophic axons is not unique to Alzheimer's disease.Am. J. Pathol. 1991; 139: 485-489PubMed Google Scholar, 24Tabaton M. Cammarata S. Mandybur T. Richey P. Kawai M. Perry G. Gambetti P. Senile plaques in cerebral amyloid angiopathy show accumulation of amyloid precursor protein without cytoskeletal abnormalities.Brain Res. 1992; 593: 299-303Crossref PubMed Scopus (13) Google Scholar), consistent with the possibility that APP may play a role in neural repair. Neurogenesis is reported to be increased in transgenic mice that overexpress APP. For example, Jin et al. (25Jin K. Galvan V. Xie L. Mao X.O. Gorostiza O.F. Bredesen D.E. Greenberg D.A. Enhanced neurogenesis in Alzheimer's disease transgenic (PDGF-APPSw,Ind) mice.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13363-13367Crossref PubMed Scopus (397) Google Scholar) reported a 2-fold increase in BrdU-labeled cells in PDGF-APPsw,Ind mice at 3 months of age. More recently, López-Toledano and Shelanski (26López-Toledano M.A. Shelanski M.L. Increased neurogenesis in young transgenic mice overexpressing human APPSw,Ind.J. Alzheimers Dis. 2007; 12: 229-240Crossref PubMed Scopus (149) Google Scholar) reported similar findings. The increase in neural precursor proliferation was attributed either to a compensatory mechanism resulting from disease pathology in the mice (25Jin K. Galvan V. Xie L. Mao X.O. Gorostiza O.F. Bredesen D.E. Greenberg D.A. Enhanced neurogenesis in Alzheimer's disease transgenic (PDGF-APPSw,Ind) mice.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13363-13367Crossref PubMed Scopus (397) Google Scholar) or to a direct effect of Aβ (26López-Toledano M.A. Shelanski M.L. Increased neurogenesis in young transgenic mice overexpressing human APPSw,Ind.J. Alzheimers Dis. 2007; 12: 229-240Crossref PubMed Scopus (149) Google Scholar). To address the role of APP in NSPC proliferation and neurogenesis, we have examined the growth and proliferation in culture of NSPCs derived from APP transgenic mice (Tg2576) and from APP knock-out (APP-KO) mice. We report that the proliferation rate of NSPCs from APP-overexpressing cells is increased and that the proliferation of NSPCs from APP-KO cells is decreased when compared with the corresponding background strain NSPCs. Furthermore, we report that this effect is mediated by a secreted factor. Despite previous suggestions that sAPPα can influence the growth of neural stem cells (9Freude K.K. Penjwini M. Davis J.L. LaFerla F.M. Blurton-Jones M. Soluble amyloid precursor protein induces rapid neural differentiation of human embryonic stem cells.J. Biol. Chem. 2011; 286: 24264-24274Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 27Lazarov O. Demars M.P. All in the family: How the APPs regulate neurogenesis.Front. Neurosci. 2012; 6: 81Crossref PubMed Scopus (55) Google Scholar, 28Hayashi Y. Kashiwagi K. Ohta J. Nakajima M. Kawashima T. Yoshikawa K. Alzheimer amyloid protein precursor enhances proliferation of neural stem cells from fetal rat brain.Biochem. Biophys. Res. Commun. 1994; 205: 936-943Crossref PubMed Scopus (60) Google Scholar), we did not find any evidence that the effect on NSPCs is mediated by sAPPα. Instead, we demonstrate that APP-induced NSPC proliferation is mediated, at least in part, by secreted cystatin C. Synthetic human sequence Aβ peptides (>95% pure) were obtained from the Keck Biotechnology Resource Laboratory (New Haven, CT). Dulbecco's modified Eagle's medium (DMEM), B27 supplement, and poly-l-lysine were from Life Technologies Australia Pty. Ltd. (Mulgrave, Australia). Penicillin, streptomycin, human recombinant sAPPα, and human recombinant EGF were all obtained from Sigma-Aldrich Pty. Ltd. (Castle Hill, Australia). Human recombinant bFGF was from PeproTech (Rocky Hill, NJ). The anti-Aβ monoclonal antibody (mAb) 6E10 was from Covance Pty. Ltd. (North Ryde, New South Wales, Australia). Mouse recombinant cystatin C, anti-mouse cystatin C antibody, affinity-purified polyclonal goat immunoglobulin G, and normal goat immunoglobulin G were from R&D Systems Inc. (Minneapolis, MN). Anti-βIII tubulin mAb was from Promega Australia (Alexandria, Australia). Normal mouse immunoglobulin G, anti-goat horseradish peroxidase-conjugated secondary antibody, and anti-mouse horseradish peroxidase-conjugated secondary antibody were from Dako Australia Pty. Ltd. (Campbellfield, Australia). Newborn pups (postnatal day 0) of APP-KO mice (from The Jackson Laboratory, Bar Harbor, ME) and their corresponding wild-type background strain controls (C57Bl/6), as well as human APP-overexpressing mice (APPSW Tg2576, from Taconic Farms Inc., Hudson, NY) and their corresponding background strain controls (C57Bl/6×SJL), were used for this study. Mice were housed in the animal facility at the University of Tasmania. All experiments were approved by the University of Tasmania Animal Ethics Committee. Primary neurosphere cultures derived from cerebral cortices of newborn mice (postnatal day 0) were prepared according to the procedure of Millet et al. (29Millet P. Lages C.S. Haïk S. Nowak E. Allemand I. Granotier C. Boussin F.D. Amyloid-β peptide triggers Fas-independent apoptosis and differentiation of neural progenitor cells.Neurobiol. Dis. 2005; 19: 57-65Crossref PubMed Scopus (34) Google Scholar). Brain cortices were cleared of meninges and hippocampus and then incubated in 1×TrypLETM Express with EDTA (Life Technologies Australia) for 10 min at 37 °C. Tissue was disrupted mechanically with a 1000-μl fine tip, and then the tissue was passed through a 40-μm cell strainer (BD Biosciences, North Ryde, Australia) to remove undissociated cells. The single neurospheres were prepared by growing cells in suspension in T75 cell culture flasks at a density of 20,000 cells/ml in proliferation medium (DMEM supplemented with 2% B27, penicillin (100 units/ml), streptomycin (100 units/ml), human bFGF (20 ng/ml), and human EGF (20 ng/ml). After 7 days in culture, neurospheres were dissociated mechanically with 200-μl fine tips, and cells were counted in a hemocytometer and then either reseeded as suspension cultures or replated as adherent cultures in 200 μl per well of proliferation medium on poly-l-lysine-coated 96-well plates at a density of 2000 cells/well. All cultures were incubated in a humidified incubator at 37 °C with 5% CO2. Cell number was measured by alamarBlue assay (28Hayashi Y. Kashiwagi K. Ohta J. Nakajima M. Kawashima T. Yoshikawa K. Alzheimer amyloid protein precursor enhances proliferation of neural stem cells from fetal rat brain.Biochem. Biophys. Res. Commun. 1994; 205: 936-943Crossref PubMed Scopus (60) Google Scholar). Dissociated cells cultured adherently on poly-l-lysine-coated 96-well plates were incubated for up to 6 days, and then 20 μl of alamarBlue reagent (Life Technologies Australia) was added into each well, and the cells were incubated for a further 4 h. The fluorescence intensity was determined using a FLUOstar Optima microplate fluorescence plate reader at an excitation wavelength of 540 nm and an emission wavelength of 590 nm. Cell number was expressed as the relative fluorescence intensity. Cell proliferation was measured by EdU incorporation. After 4 days in proliferation medium, cells were incubated with EdU for 8 h as described previously (30Young K.M. Psachoulia K. Tripathi R.B. Dunn S.J. Cossell L. Attwell D. Tohyama K. Richardson W.D. Oligodendrocyte dynamics in the healthy adult CNS: evidence for myelin remodeling.Neuron. 2013; 77: 873-885Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). Neurospheres were mechanically dissociated, and then isolated cells were plated at a density of 105 cells/well in 24-well plate. The cells were grown in a differentiation medium (DMEM containing 1% fetal calf serum (FCS), 2% B27 supplement, 100 units/ml penicillin, and 100 units/ml streptomycin) for 5 days at 37 °C in an atmosphere containing 5% CO2. The cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) (8 g/liter NaCl, 0.2 g/liter KCl, 1.44 g/liter Na2HPO4, and 0.24 g/liter KH2PO4, pH 7.2) for 15 min, permeabilized with 0.03% (v/v) Triton X-100 in PBS for 5 min, and then blocked in 4% goat serum in PBS for 20 min. Fixed cells were stained with mouse anti-βIII tubulin antibody (1:1000 diluted in 2% goat serum in PBS) and then incubated with a goat anti-mouse IgG conjugated to Alexa Fluor 488 (1:1000 diluted in 2% goat serum in PBS) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) at 1:7000 dilution. Conditioned medium was collected from neurosphere cultures that had been grown over a period of 7 days. To examine the effect of conditioned medium on NSPC proliferation, dissociated cells from neurospheres were cultured adherently on poly-l-lysine-coated 96-well plates in 100 μl/well of proliferation medium. Sixteen hours after plating, 100 μl of conditioned medium or normal proliferation medium (control) was added, and the cells were incubated for 3 or 5 days, after which proliferation was measured using the alamarBlue assay. The level of sAPPα and cystatin C in conditioned medium was determined by Western blotting. The volume per well of conditioned medium that was analyzed was adjusted so that it represented the same number of viable cells, as determined using the alamarBlue assay. Routinely, ∼10–30 μl was loaded into each gel lane for analysis. For the analysis of intracellular cystatin C, cells were washed with PBS and then lysed as described previously (31Cui H. Hung A.C. Klaver D.W. Suzuki T. Freeman C. Narkowicz C. Jacobson G.A. Small D.H. Effects of heparin and enoxaparin on APP processing and Aβ production in primary cortical neurons from Tg2576 mice.PLoS One. 2011; 6: e23007Crossref PubMed Scopus (25) Google Scholar) prior to analysis by Western blotting. Proteins were separated on 8% (sAPPα) or 12% (cystatin C) sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels before being transferred electrophoretically onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for 1 h with 5% skim milk powder in 50 mm Tris-buffered saline (pH 8) containing 0.05% Tween 20 (TBS-Tween) and incubated overnight at 4 °C with either anti-APP antibody (22C11 at 1:1000 dilution or 6E10 at 1:1000 dilution) or anti-cystatin C antibody (1:1000 dilution). Chemiluminescence reactions were monitored using a CHEMI-SMART 5000, and images were collected using Chemi-Capt 50001. For quantification of immunoreactivity, images of blots were analyzed using ImageJ version 1.46. For depletion of sAPPα or cystatin C from conditioned medium, mAb 6E10, anti-mouse cystatin C antibody, or goat immunoglobulin G (35 μg) was incubated with 500 μl of protein G agarose gel (Roche Products Pty. Ltd., Dee Why, Australia) overnight at 4 °C in 5 ml of PBS. The gel was then washed three times with 5 ml PBS, after which the gel was incubated with 5.5 ml of conditioned medium for 3 h at 4 °C. Finally, the gel slurry containing conditioned medium was centrifuged (10,000 × g), and the resulting supernatant fraction was assayed by Western blotting or used for cell proliferation experiments. RNA was extracted from the neurospheres of n = 6 independent mouse cohorts using an RNeasy mini kit (Qiagen Pty. Ltd., Chadstone, Australia) as described by the manufacturer. Each preparation of neurospheres contained ∼106 cells. Six independent RNA extracts were obtained from each neurosphere preparation. cDNA was obtained from 400 ng of RNA with an RT2 First Strand kit (Qiagen) as described by the manufacturer. The cystatin C primers (Cst3) were from Qiagen, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were from Geneworks Pty. Ltd. (Hindmarsh, Australia). GAPDH was used as an internal control. All primers were used at a concentration of 10 μm. All samples were diluted 1:10 and analyzed in triplicate. Standard curves for Cst3 and GAPDH with concentrations 1, 0.5, 0.25, and 0.125 μg were used to quantify Cst3 mRNA using SYBR master mix (Qiagen). The results were analyzed using a LightCycler 480 (Roche Diagnostics Australia Pty. Ltd., Castle Hill, Australia). Statistical analysis was performed with GraphPad Prism software, version 5.04. Data were tested by one-way ANOVA or Student's t test. Post hoc comparisons were analyzed using Tukey's test. Differences were considered statistically significant when p < 0.05. Data values are presented as the means ± S.E. All results were derived from at least three independent experiments in which cells were derived from at least three different mice of the same strain. We first examined the level of APP in neurosphere cultures to confirm that the level of APP expression was higher in the Tg2576 cultures than in the background strain (C57Bl/6xSJL) littermate control cultures. At 2, 4, and 6 days after plating, conditioned medium was analyzed for APP by Western blotting with mAb 22C11,which recognizes both mouse and human APP and the APP homologue amyloid protein-like protein-2, and with mAb 6E10, which recognizes human sAPPα. The results confirmed that APP levels were much higher in the medium of Tg2576 neurosphere cultures than in the background strain cultures (Fig. 1A). A major band of 100–110 kDa was detected in the medium, corresponding to the molecular mass of sAPPα. Next, to examine the role of APP in NSPC proliferation, we compared the proliferation of cells derived from Tg2576 mice with that of the background strain controls. Neurosphere cultures were dissociated into a single cell suspension on day 7, and then cells were cultured adherently on poly-l-lysine-coated 96-well plates. The proliferation of the cells was measured using an alamarBlue assay. Fluorescence intensity in an alamarBlue assay was taken as an index of the number of viable cells. Overall, the growth rate of the cells derived from the Tg2576 mice was significantly greater (p < 0.05, one-way ANOVA with post hoc Tukey's test) than that of the cells derived from the background strain mice (Fig. 1B). We also examined the growth of neurospheres to determine whether the proliferation of NSPCs was increased in the Tg2576 cultures. Neurospheres were cultured for a period of 7 days, after which they were examined under phase-contrast microscopy (Fig. 1, C and D). Neurospheres derived from Tg2576 mice were on average greater in size than the neurospheres from the background strain mice, confirming that the growth of NSPCs in Tg2576 cultures was greater than that of the background strain cultures. To confirm that the increased growth of the Tg2576 NSPC was due to a higher proliferation rate, we measured proliferation using an EdU uptake assay (30Young K.M. Psachoulia K. Tripathi R.B. Dunn S.J. Cossell L. Attwell D. Tohyama K. Richardson W.D. Oligodendrocyte dynamics in the healthy adult CNS: evidence for myelin remodeling.Neuron. 2013; 77: 873-885Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). The percentage of EdU-positive cells in the Tg2576 cultures was ∼20% higher than the cultures derived from background strain mice (Fig. 1E). These results clearly supported the view that the increased growth observed using the alamarBlue assay and in the neurosphere cultures was due to an increase in the amount of NSPC proliferation. To determine whether the higher proliferation rate of the Tg2576 cells was also associated with a higher potential for neuronal differentiation, isolated NSPCs, grown adherently, were transferred to a differentiation medium containing 1% FCS but lacking EGF and bFGF. The cells were incubated over 5 days and then immunostained for the neuronal marker βIII-tubulin (Fig. 2). The results showed that there was an increased proportion of neurons in the Tg2576 cell population when compared with the cells derived from the background strain (C57Bl/6xSJL) mice (Fig. 2). Tg2576 cells also possessed longer, more extensive neurite networks than the background strain cells. To examine whether expression of endogenous mouse APP influences NSPC proliferation and to rule out the possibility that the increase in proliferation observed in Tg2576 cultures was due to a factor unrelated to APP overexpression, we also examined the proliferation rate of NSPCs from APP-KO mice. We found that NSPC proliferation was decreased in APP-KO cultures when compared with the corresponding background strain (C57Bl/6) cultures (Fig. 3). The growth rate of the APP-KO cells, as assessed by an alamarBlue assay, was ∼60% of that of the cells derived from background strain mice (Fig. 3A). Furthermore, neurospheres from APP-KO mice were smaller and less numerous than the C57Bl/6 neurospheres (Fig. 3, B and C). In addition, the percentage of proliferating EdU-positive cells was also significantly decreased. These experiments clearly demonstrated that endogenous APP was also involved in the regulation of NSPC proliferation. We also examined the capacity of APP-KO NSPCs to differentiate using the same procedure as described previously for the Tg2576 cells. The APP-KO cells were compared with the corresponding C57Bl/6 background strain cells. The total number of βIII-tubulin-positive cells was lower in the APP-KO cultures than in the background strain cultures (Fig. 4). We next examined whether the effect of APP on proliferation was mediated by a factor that was secreted into the culture medium. Conditioned medium was prepared over 7 days from Tg2576 and the corresponding background strain (C57Bl/6xSJL) neurosphere cultures. In parallel, dissociated C57Bl/6xSJL NSPCs were plated and incubated for 16 h. Conditioned medium from the neurosphere cultures or unconditioned proliferation medium was then added to the dissociated cell cultures. The amount of proliferation was measured after 5 days of incubation. There was a higher rate of proliferation in cultures containing conditioned medium (whether from C57Bl/6xSJL or Tg2576 cell" @default.
• W1993011380 created "2016-06-24" @default.
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• W1993011380 date "2013-06-01" @default.
• W1993011380 modified "2023-10-11" @default.
• W1993011380 title "Role of Cystatin C in Amyloid Precursor Protein-induced Proliferation of Neural Stem/Progenitor Cells" @default.
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