Role of amyloid precursor protein in neural stem/progenitor cell proliferation and differentiation

摘要

Alzheimer’s disease (AD) is a progressive neurodegenerative condition thatudcommonly affects people over the age of 65. There are currently no effectiveudtreatments which prevent or delay the progression of AD. The disease is characterizedudby two major pathological hallmarks in the brain, extracellular amyloid plaques andudintracellular neurofibrillary tangles.udThe β-amyloid protein (Aβ) is the major component of the amyloid plaques and isudconsidered to play a central role in AD pathogenesis. Aβ is produced by proteolyticudprocessing of the amyloid precursor protein (APP). However, the normal function ofudAPP remains unclear despite numerous studies. Understanding the biological functionudof APP may improve our understanding into the molecular basis of AD pathology.udAPP expression has been reported to increase in neurons during embryogenesis. Inudaddition, enhanced neurogenesis has been observed in the brains of AD patients andudin transgenic mice which overexpress APP. However, other studies have reportedudimpaired neurogenesis in APP transgenic mice. Thus, the studies presented here wereudaimed at investigating the role of APP in neural stem/progenitor cell (NSPC)udproliferation and differentiation.udTo test the effect of APP on NSPC proliferation, NSPCs derived from human APPudoverexpressing transgenic (Tg2576) mice and APP knockout (APP KO) mice andudtheir corresponding background strain wild type (WT) mice were cultured in audproliferation medium containing growth factors and NSPC growth was measured. Theudstudy found than Tg2576 NSPCs proliferated more rapidly than NSPCs from WT control mice. In contrast, NSPCs derived from APP KO mice proliferated less readilyudthan the corresponding background strain mice.udThe secreted fragments of APP, Aβ and sAPPα, have been reported to affectudneurogenesis. The molecular basis of the effect is uncertain. To examine whetherudsAPPα or two major isoforms of Aβ, Aβ40 and Aβ42, were responsible for APP -udinduced NSPC proliferation, the WT NSPCs were grown in proliferation mediumudcontaining sAPPα, Aβ40 or Aβ42 and the cell proliferation was measured by examiningudcell viability. No significant difference in cell viability was found between the non –udtreatment groups and the groups treated with sAPPα, Aβ40 or Aβ42. Besides,udimmunodepletion of secreted fragments of APP (sAPPα, Aβ) from Tg2576udconditioned medium did not lower NSPC proliferation, indicating that neither sAPPαudnor Aβ contributed to the proliferation effect.udTo examine whether other secreted factors might be involved in the proliferationudeffect, the ability of cell conditioned medium to stimulate proliferation was tested.udNSPC conditioned medium from Tg2576 cultures was found to increase proliferationudwhile conditioned medium from APP KO cultures was found to have a lower effectudon proliferation. The effect on proliferation was found to be due to a secreted factor,udcystatin C (CysC), which has previously been reported to promote NSPCudproliferation. Immunodepletion of CysC from the Tg2576 conditioned mediumudremoved the stimulatory effect of APP on NSPC proliferation. mRNA levels of CysCudin APP KO cells were lower than the wild-type control cells. Therefore, CysC isudlikely to be a major mediator of APP-associated NSPC proliferation.udCysC is well characterized as a cysteine protease inhibitor. Proteases have beenudreported to play a role on cell proliferation, thus regulation of proteases activities mayudaffect NSPC proliferation. To examine whether the effect of CysC on NSPCudproliferation was due to an effect on one or more cysteine proteases, NSPCs wereudtreated with the broad spectrum protease inhibitor, chymostatin, the specific cysteineudprotease inhibitor, E-64, the aspartic protease inhibitor, pepstatin A and the serine andudcysteine protease inhibitor, antipain, after which cell proliferation was examined. Theudcysteine protease inhibitor, E-64 and the serine/cysteine protease inhibitor, antipain,udboth of which are cell membrane impermeable were found to stimulate NSPCudproliferation at low concentrations. However, no extracellular cysteine proteases wereudidentified bound to CysC in the conditioned medium by the CysC affinityudchromatography. Thus more studies may be required in the future to identify theudspecific target of CysC.udTo test the effect of APP on NSPC differentiation, NSPCs prepared from Tg2576, andudAPP KO and corresponding background strain wild type mice were cultured in auddifferentiation medium lacking of growth factors. Cultures were immunostained forudmarkers of neurons (βIII tubulin), astrocytes (GFAP) and oligodendrocyte progenitorsud(NG2) various days after incubation to assess cell differentiation. APP overexpressingud(Tg2576) NSPC cultures displayed more βIII tubulin+ and GFAP+ cells than the wildudtype cultures, while APP KO NSPC cultures had fewer βIII tubulin+ and GFAP+ cellsudcompared to the corresponding wild type cultures. No significant difference wasudfound in NG2 expression.udPreviously published studies on the effect of sAPPα, Aβ40 and Aβ42 on NSPCuddifferentiation have yielded conflicting results. Therefore, one of the aims of thisudstudy was to examine whether CysC, sAPPα, Aβ40 and Aβ42 influence NSPCuddifferentiation. The APP KO NSPCs were grown in differentiation mediumudcontaining CysC, sAPPα, Aβ40 and Aβ42, and the cells were fixed and immunostainedudfor markers of neurons (βIII tubulin), astrocytes (GFAP) and oligodendrocyteudprogenitors (NG2). No difference in the expression of the neuronal and glial markersudbetween the control group and groups treated with CysC, sAPPα, Aβ40 and Aβ42 wasudobserved. It was concluded that neither CysC nor sAPPα, Aβ influence NSPCuddifferentiation.udIn summary, the study found that APP can promote NSPC proliferation in vitro, andudthe effect is mediated by secreted CysC. However, the molecular basis of CysC actionudrequires further studies. In addition, APP was also found to play a role on NSPCuddifferentiation in vitro. Adult neurogenesis has been reported to play important rolesudin learning, memory and mood regulation. Therefore, therapeutic strategies focusingudon anti – Aβ production by alteration APP processing should be considered carefully.udBesides, APP - directed neuronal differentiation of NSPCs could perhaps contributeudto stem cell therapy for neurodegenerative disease treatments including AD.