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

Alzheimer’s disease (AD) is a progressive neurodegenerative condition that commonly affects people over the age of 65. There are currently no effective treatments which prevent or delay the progression of AD. The disease is characterized by two major pathological hallmarks in the brain, extracellular amyloid plaques and intracellular neurofibrillary tangles. The β-amyloid protein (Aβ) is the major component of the amyloid plaques and is considered to play a central role in AD pathogenesis. Aβ is produced by proteolytic processing of the amyloid precursor protein (APP). However, the normal function of APP remains unclear despite numerous studies. Understanding the biological function of APP may improve our understanding into the molecular basis of AD pathology. APP expression has been reported to increase in neurons during embryogenesis. In addition, enhanced neurogenesis has been observed in the brains of AD patients and in transgenic mice which overexpress APP. However, other studies have reported impaired neurogenesis in APP transgenic mice. Thus, the studies presented here were aimed at investigating the role of APP in neural stem/progenitor cell (NSPC) proliferation and differentiation. To test the effect of APP on NSPC proliferation, NSPCs derived from human APP overexpressing transgenic (Tg2576) mice and APP knockout (APP KO) mice and their corresponding background strain wild type (WT) mice were cultured in a proliferation medium containing growth factors and NSPC growth was measured. The study found than Tg2576 NSPCs proliferated more rapidly than NSPCs from WT control mice. In contrast, NSPCs derived from APP KO mice proliferated less readily than the corresponding background strain mice. The secreted fragments of APP, Aβ and sAPPα, have been reported to affect neurogenesis. The molecular basis of the effect is uncertain. To examine whether sAPPα or two major isoforms of Aβ, Aβ40 and Aβ42, were responsible for APP - induced NSPC proliferation, the WT NSPCs were grown in proliferation medium containing sAPPα, Aβ40 or Aβ42 and the cell proliferation was measured by examining cell viability. No significant difference in cell viability was found between the non – treatment groups and the groups treated with sAPPα, Aβ40 or Aβ42. Besides, immunodepletion of secreted fragments of APP (sAPPα, Aβ) from Tg2576 conditioned medium did not lower NSPC proliferation, indicating that neither sAPPα nor Aβ contributed to the proliferation effect. To examine whether other secreted factors might be involved in the proliferation effect, the ability of cell conditioned medium to stimulate proliferation was tested. NSPC conditioned medium from Tg2576 cultures was found to increase proliferation while conditioned medium from APP KO cultures was found to have a lower effect on proliferation. The effect on proliferation was found to be due to a secreted factor, cystatin C (CysC), which has previously been reported to promote NSPC proliferation. Immunodepletion of CysC from the Tg2576 conditioned medium removed the stimulatory effect of APP on NSPC proliferation. mRNA levels of CysC in APP KO cells were lower than the wild-type control cells. Therefore, CysC is likely to be a major mediator of APP-associated NSPC proliferation. CysC is well characterized as a cysteine protease inhibitor. Proteases have been reported to play a role on cell proliferation, thus regulation of proteases activities may affect NSPC proliferation. To examine whether the effect of CysC on NSPC proliferation was due to an effect on one or more cysteine proteases, NSPCs were treated with the broad spectrum protease inhibitor, chymostatin, the specific cysteine protease inhibitor, E-64, the aspartic protease inhibitor, pepstatin A and the serine and cysteine protease inhibitor, antipain, after which cell proliferation was examined. The cysteine protease inhibitor, E-64 and the serine/cysteine protease inhibitor, antipain, both of which are cell membrane impermeable were found to stimulate NSPC proliferation at low concentrations. However, no extracellular cysteine proteases were identified bound to CysC in the conditioned medium by the CysC affinity chromatography. Thus more studies may be required in the future to identify the specific target of CysC. To test the effect of APP on NSPC differentiation, NSPCs prepared from Tg2576, and APP KO and corresponding background strain wild type mice were cultured in a differentiation medium lacking of growth factors. Cultures were immunostained for markers of neurons (βIII tubulin), astrocytes (GFAP) and oligodendrocyte progenitors (NG2) various days after incubation to assess cell differentiation. APP overexpressing (Tg2576) NSPC cultures displayed more βIII tubulin+ and GFAP+ cells than the wild type cultures, while APP KO NSPC cultures had fewer βIII tubulin+ and GFAP+ cells compared to the corresponding wild type cultures. No significant difference was found in NG2 expression. Previously published studies on the effect of sAPPα, Aβ40 and Aβ42 on NSPC differentiation have yielded conflicting results. Therefore, one of the aims of this study was to examine whether CysC, sAPPα, Aβ40 and Aβ42 influence NSPC differentiation. The APP KO NSPCs were grown in differentiation medium containing CysC, sAPPα, Aβ40 and Aβ42, and the cells were fixed and immunostained for markers of neurons (βIII tubulin), astrocytes (GFAP) and oligodendrocyte progenitors (NG2). No difference in the expression of the neuronal and glial markers between the control group and groups treated with CysC, sAPPα, Aβ40 and Aβ42 was observed. It was concluded that neither CysC nor sAPPα, Aβ influence NSPC differentiation. In summary, the study found that APP can promote NSPC proliferation in vitro, and the effect is mediated by secreted CysC. However, the molecular basis of CysC action requires further studies. In addition, APP was also found to play a role on NSPC differentiation in vitro. Adult neurogenesis has been reported to play important roles in learning, memory and mood regulation. Therefore, therapeutic strategies focusing on anti – Aβ production by alteration APP processing should be considered carefully. Besides, APP - directed neuronal differentiation of NSPCs could perhaps contribute to stem cell therapy for neurodegenerative disease treatments including AD