Neural stem cells isolated from amyloid precursor protein-mutated mice for drug discovery

Abstract

Aim: To develop an in vitro model based on neural stem cells derived from transgenic animals, to be used in the study of pathological mechanisms of Alzheimer's disease and for testing new molecules.

Methods: Neural stem cells (NSCs) were isolated from the subventricular zone of Wild type (Wt) and Tg2576 mice. Primary and secondary neurosphere generation was studied, analysing population doubling and the cell yield per animal. Secondary neurospheres were dissociated and plated on MCM Gel Cultrex 2D and after 6 d in vitro (DIVs) in mitogen withdrawal conditions, spontaneous differentiation was studied using specific neural markers (MAP2 and TuJ-1 for neurons, GFAP for astroglial cells and CNPase for oligodendrocytes). Gene expression pathways were analysed in secondary neurospheres, using the QIAGEN PCR array for neurogenesis, comparing the Tg2576 derived cell expression with the Wt cells. Proteins encoded by the altered genes were clustered using STRING web software.

Results: As revealed by 6E10 positive staining, all Tg2576 derived cells retain the expression of the human transgenic Amyloid Precursor Protein. Tg2576 derived primary neurospheres show a decrease in population doubling. Morphological analysis of differentiated NSCs reveals a decrease in MAP2- and an increase in GFAP-positive cells in Tg2576 derived cells. Analysing the branching of TuJ-1 positive cells, a clear decrease in neurite number and length is observed in Tg2576 cells. The gene expression neurogenesis pathway revealed 11 altered genes in Tg2576 NSCs compared to Wt.

Conclusion: Tg2576 NSCs represent an appropriate AD in vitro model resembling some cellular alterations observed in vivo, both as stem and differentiated cells.

Keywords: Alzheimer’s disease; Drug discovery; Neural stem cells; Neuron maturation.

Figure 1

Transgenic protein expression and population doubling. Micrograph shows the positive 6E10 staining of Tg2576 plated cells derived from secondary neurospheres (A) and the related negative staining of wild type derived cells (B). A decrease in population doubling seems to occur in the first few days of primary neurosphere development (C), and is not repeated in secondary neurosphere formation (D). Statistical analysis: Student’s t-test. Graphs represent mean ± SE and asterisks represent significant differences between Tg2576 and Wt at the same DIV (^aP < 0.05).

Figure 2

Morphological analysis. A-D: Micrographs showing immunostaining for β-III-tubulin (TuJ-1) and MAP2 in Wt and Tg2576 cells. Hoechst 33258 nuclear staining was used to determine the total cell number; E: The analyses of cell lineage indicate an increase in GFAP- and a decrease in MAP2-positive cells in Tg2576 compared to Wt. The number of CNPase-positive cells does not vary between the two genotypes; F, G: neural maturation analysis shows both a decrease in TuJ-1 positive branching of the third order and in neurite extension in cells derived from Tg2576 animals. Statistical analysis: Student’s t-test. Graphs represent mean ± SE and asterisks represent significant differences between Tg2576 and Wt (^aP < 0.05; ^bP < 0.01; ^dP < 0.001). Bars: A, C: 100 μm; B, D, 50 μm.

Figure 3

Functional protein net. The list of the neurogenesis-involved genes altered in Tg2576 versus Wt cells is reported in Table 2. The related proteins were clustered using STRING web software. Figure shows the 11 genes (A) and the extended net (B).