The prion protein regulates beta-amyloid-mediated self-renewal of neural stem cells in vitro
© Collins et al.; licensee BioMed Central. 2015
Received: 23 October 2014
Accepted: 25 March 2015
Published: 11 April 2015
The beta-amyloid (Aβ) peptide and the Aβ-oligomer receptor, prion protein (PrP), both influence neurogenesis. Using in vitro murine neural stem cells (NSCs), we investigated whether Aβ and PrP interact to modify neurogenesis. Aβ imparted PrP-dependent changes on NSC self-renewal, with PrP-ablated and wild-type NSCs displaying increased and decreased cell growth, respectively. In contrast, differentiation of Aβ-treated NSCs into mature cells was unaffected by PrP expression. Such marked PrP-dependent differences in NSC growth responses to Aβ provides further evidence of biologically significant interactions between these two factors and an important new insight into regulation of NSC self-renewal in vivo.
Alzheimer’s disease (AD) is the most common form of dementia. The core components of the senile plaques that characterise AD pathologically are beta-amyloid (Aβ) peptides cleaved from the amyloid precursor protein (APP). Various Aβ species exist as a result of differing N- and C-terminal processing sites and these species can aggregate, forming oligomers that are implicated in Aβ toxicity . Most Aβ species are found in healthy brain tissue but the relative amounts shift during AD [2,3]. In health, Aβ1-40 predominates and during AD Aβ1-42, Aβ4-42 and pyroglutamated Aβ3-42 (3(pE)-42) are increased . Many other species also exist with their relative amounts changing during disease.
Neurogenesis, whilst declining significantly in the adult organism, continues throughout life. Adult neural stem cells (NSCs) are confined to specific protected sites within the brain, including the sub-granular zone (SGZ) of the dentate gyrus and sub-ventricular zone of the lateral ventricle . Adult NSCs can self-renew and are multipotent; they can differentiate into cells of any central nervous system lineage. In the brains of AD patients, markers of neurogenesis are increased [5,6] indicating potential neurogenic dysregulation or stimulated compensation for neuronal loss. AD pathology typically begins in the transentorhinal and entorhinal cortex . This region lies adjacent to the SGZ and, therefore, NSCs in their normally protected niche environment may be exposed to hostile conditions that stimulate them to change their behaviour . There is significant evidence that Aβ peptides are able to modulate neurogenesis [8-10]. Various discrepancies exist in the literature as to whether neurogenesis is enhanced or suppressed by Aβ exposure, which is most likely due to the manner in which the Aβ was prepared (that is, if monomeric, oligomeric or fibrillar Aβ species were used) and the model system for NSC study (for example, in vivo, in vitro, mouse strain); however, the consensus is in favour of changed NSC behaviour following exposure to Aβ species.
Neurogenesis is also modulated by another neurodegenerative disease-associated protein, the prion protein (PrP) [11,12]. Increased PrP expression is associated with increased cell cycling at the expense of differentiation . Recent studies found that PrP is an essential receptor that transduces soluble Aβ1-42 oligomer signals from the plasma-membrane through the NMDA receptor via the signalling molecule fyn to tau, with this signalling thought to cause cellular toxicity [14-16]. Based on the knowledge that both Aβ and PrP can individually modulate neurogenesis and that PrP is a soluble Aβ1-42 binding partner necessary for the transduction of toxic signals, we hypothesized that PrP might also transduce the Aβ peptide signals that alter neurogenesis. The present study therefore investigated the ability of various Aβ peptides to modulate in vitro self-renewal and differentiation of adult NSCs harvested from PrP gene-ablated (knock-out (KO)) or from wild-type (WT; normal PrP expression) mice.
Aβ-amyloid peptides (China Peptides, China) were prepared as described previously . NSC harvest and routine culture was as described previously [18,19]. For the neural colony-forming assay, cells were seeded in a semi-solid gel matrix made with a 2:1 solution of proliferation medium and collagen. After day 21, neurospheres were counted and their diameter measured using NIS-Elements (Nikon Adelaide, Australia) software. Cell cycle analysis was performed using the Muse Cell Cycle Kit (Millipore, Bayswater, Victoria AUS). For plate and blotting assays cells were cultured as an adherent monolayer on a 1:1 poly-D-lysine-laminin matrix. Cellular ATP content was measured using Life Sciences’ ATP assay (Invitrogen, Mulgrave, Victoria AUS). Immunodetection methods have been described previously [18-20]. Expanded methodology is provided in Additional file 1.
Results and discussion
Potential Aβ-PrP signalled changes in neurogenesis were assessed using four Aβ species; Aβ1-40, Aβ1-42, Aβ4-42 and Aβ3(pE)-42, representing those that are found ‘normally’ in health and those that have been linked with cellular toxicity in AD [21,22]. Previous studies have shown that fibrillar Aβ has no effect on neurogenesis  and soluble oligomeric Aβ42 is toxic; therefore, Aβ peptides were prepared using an established protocol for producing soluble monomeric species . One μM Aβ peptide was used based on results of previous studies that demonstrated this concentration induced neurogenic effects . No toxicity was observed at this concentration throughout the duration of the assays (Additional file 2).
To further assess differences in NSC growth, Aβ1-42 was used to assess the number of cells in each phase of the cell cycle 24 hours post-Aβ addition to liquid culture. More KO cells rested in G0/G1 basally than WT (two-way ANOVA, F = 22.99, P = 0.003, n = 4) but after treatment with Aβ the number of KO cells actively cycling (S, G2/M phases) was significantly increased (two-way ANOVA for S phase, F = 39.18, P = 0.003, n = 4; for G2/M phases, F = 24.91, P = 0.003, n = 4; Figure 1F). There was no significant change for the WT cells, suggesting that the changes that slow the growth and reduce the diameter of neurospheres may occur after a longer period of exposure to Aβ.
Changes in the rate of cell cycling are likely to require energy. Therefore, markers of cellular metabolism and mitochondrial function, as well as cell cycle, were compared in WT and KO NSCs following Aβ treatment. Formazan metabolism was significantly increased in NSC KO cells compared with the WT cells when cells were treated with Aβ1-40 and Aβ4-42 (two-way ANOVA, F = 16.55, P < 0.001, n = 3, see Additional file 5 for basal data and Additional file 7 for full statistical analyses; Figure 3B). Cellular ATP levels were generally unchanged between the KO and WT NSCs, only showing a decrease in WT cells treated with Aβ4-42 (two-way ANOVA, F = 5.95, P = 0.021, n = 4; Figure 3C). This steady-state ATP measurement does not preclude increased production balanced by increased use. Contrary to decreased growth, TOMM22, a mitochondrial outer membrane translocase, was increased in WT cells when treated with Aβ1-40, Aβ4-42, and Aβ3(pE)-42 (two-way ANOVA, F = 16.45, P < 0.001, n = 3; Figure 3D,E). Potentially, this might indicate that Aβ can exert an effect on mitochondria signalled through PrP that could be detrimental to their function, thus limiting growth. The cell cycle marker Pin1 has also been shown to protect against tau hyperphosphorylation and subsequent changes to the cellular cytoskeleton . Pin1 was globally increased in the Aβ-treated WT compared with the KO cells, although only the Aβ4-42 condition was individually significant (two-way ANOVA, F = 21.98, P < 0.001, n = 4; Figure 3F,G). The overall increase in Pin1 could reflect a failed effort to increase cell cycling in these cells or might represent a cellular protective response against Aβ. p53 is linked with cell cycle and also with cell death. When activated, the half-life of this protein increases resulting in increased protein detection over time. No changes in p53 protein were observed upon treatment with Aβ in either the KO or WT NSCs (Figure 3H). We additionally considered other previously PrP-linked Aβ signalling pathways (fyn, GSK-3β and calcium) finding no changes in Aβ-induced responses that relate to PrP expression (Additional file 8).
When the localisation of Aβ added to cells was considered relative to PrP, surface staining of Aβ was observed to be more intense on the WT cells after 1 hour and PrP surface staining appeared less with greater signal inside the cells (Figure 3I). These findings are consistent with previous studies that have shown Aβ preferentially binds to cells expressing PrP causing internalisation  and also supports the hypothesis that Aβ stimulates different pathways in KO and WT cells.
It remains to be determined how NSCs are affected by PrP-linked Aβ signalling in vivo, where the context of their support cells and scaffold may lead to more diverse outcomes. However, the clear responses of the cells cultured in vitro indicates that sufficient cellular machinery and environmental factors to transduce PrP-Aβ signalling cascades are present. The details of these pathways will be revealed by future investigation, but it is of interest that the classical Aβ signalling pathways evaluated here were unaffected. Hypothetically, as PrP-Aβ studies to date were performed in neuronal cultures or mice, different pathways could be engaged in NSCs and post-mitotic neurones or, alternatively, differing pathways may be activated when Aβ levels reach toxic concentrations. Furthermore, during sporadic AD, brain PrP expression reduces , inversely correlating with Aβ burden. Reduced PrP expression (akin to KO NSCs) may permit Aβ stimulation of NSC proliferation, resulting in the increased neurogenic markers seen in AD brain tissue [5,6].
analysis of variance
neural stem cell
The authors would like to acknowledge Professor Colin Masters for his ongoing support and thank Dr Theo Mantamadiotis and Ms Gulay Filiz for their assistance with the cell cycle flow cytometry. This work was supported by an NHMRC program grant (#628946) and the Victorian Government’s Operational Infrastructure Support program. SJC is supported by an NHMRC Practitioner Fellowship (#APP100581), SCD by an ARC Future Fellowship (FT110100199) and TMR by an Alzheimer’s Australia fellowship.
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