Presenilin 1 mutants impair the self-renewal and differentiation of adult murine subventricular zone-neuronal progenitors via cell-autonomous mechanisms involving notch signaling

Abstract

The vast majority of pedigrees with familial Alzheimer's disease (FAD) are caused by inheritance of mutations in the PSEN1 1 gene. While genetic ablation studies have revealed a role for presenilin 1 (PS1) in embryonic neurogenesis, little information has emerged regarding the potential effects of FAD-linked PS1 variants on proliferation, self-renewal and differentiation, key events that control cell fate commitment of adult brain neural progenitors (NPCs). We used adult brain subventricular zone (SVZ)-derived NPC cultures transduced with recombinant lentivirus as a means to investigate the effects of various PS1 mutants on self-renewal and differentiation properties. We now show that viral expression of several PS1 mutants in NPCs leads to impaired self-renewal and altered differentiation toward neuronal lineage, in vitro. In line with these observations, diminished constitutive proliferation and steady-state SVZ progenitor pool size was observed in vivo in transgenic mice expressing the PS1DeltaE9 variant. Moreover, NPC cultures established from the SVZ of adult mice expressing PS1DeltaE9 exhibit reduced self-renewal capacity and premature exit toward neuronal fates. To these findings, we show that both the levels of endogenous Notch/CBF-1-transcriptional activity and transcripts encoding Notch target genes are diminished in SVZ NPCs expressing PS1DeltaE9. The deficits in self-renewal and multipotency are restored by expression of Notch1-ICD or a downstream target of the Notch pathway, Hes1. Hence, we argue that a partial reduction in PS-dependent gamma-secretase processing of the Notch, at least in part, accounts for the impairments observed in SVZ NPCs expressing the FAD-linked PS1DeltaE9 variant.

Figure 1.

Self-renewal capacity of NPCs expressing FAD-linked PS1 mutants. A, NPCs grown as neurospheres from dissected SVZ of adult mice in serum-free medium conditions (i). Scale bar, 50 μm. Immunoreactivity of neurosphere for NPC marker antigens, nestin (ii; green) and GFAP (iv; red). Nuclei were stained with DAPI (iii, v; blue). Scale bar, 25 μm. Lineage committed progeny cells with distinct immunoreactivity to βIII-tubulin (vi; green, thick arrow) or GFAP (vii; red, thin arrow) were observed following differentiation of NPCs and the overlay of (vi) and (vii) is shown in viii. Scale bar, 10 μm. B, Illustration of lentiviral vector constructs encoding cDNAs for PShWT or PS1 variants and copGFP driven under the control of two independent promoters. C, Total protein lysate of PS-deficient fibroblasts transduced with mentioned lentivirus were Tris-Tricine SDS-PAGE resolved and immunoblotted with either human PS1 N-terminal fragment (NTF) or GFP antisera. Protein (25 μg) was loaded in each lane. D, LV-transduced NPCs that self-renewed as neurospheres under serum-free conditions expressed GFP (−FBS). Left and the middle panel on the top row show the bright-field and fluorescent images of LV-transduced neurospheres under 10× objective, respectively (Scale bar: 50 μm). Magnified image of a copGFP⁺ neurosphere captured under 20× objective is shown in the panel on right (top row). LV-transduced progenitors were tracked in FBS-treated, differentiated cultures based on the expression of GFP (+FBS, bottom row; bright-field and the respective fluorescent image of transduced progenitor cells; scale bar, 10 μm). Tracing of infected cells by GFP expression was used to evaluate the effect of PS1 mutants on self-renewal and differentiation of adult SVZ NPCs. E, Self-renewal capacity of GFP⁺ NPCs (neurosphere, NS) transduced with lentivirus encoding PShWT or various FAD-PS1 mutant cDNA. Data represent m ± SEM obtained from 6 independent experiments. *p < 0.05 for NPCs transduced with LV-PS1hWT vs LV-PS1 mutants.

Figure 2.

Multilineage potential of NPCs expressing FAD-linked PS1 mutants. A, Representative photomicrographs reveal the identity of LV-PS1-transduced, GFP⁺ NPCs (arrow), differentiated into βIII-tubulin (row i), MAP2 (row ii), GFAP (row iii) or s100β (row iv) lineages. Overlay of the respective images are shown on the right of each row. Scale bar, 25 μm. B–E, Quantification of GFP⁺ progenitors differentiated to neuronal or astroglial progeny cell types. Data represent m ± SEM obtained from 3 independent experiments and the differentiated fate was scored by counting a minimum of 100 GFP⁺ cells that colabeled with neuronal or astrocyte cell fate marker. *p < 0.05; **p < 0.01, for NPCs transduced with LV-PS1hWT vs LV-PS1 variants.

Figure 3.

Steady-state levels of progenitor pool in postnatal SVZ of PS1ΔE9 transgenic mice. A, Confocal images of BrdU-labeled cells (green) in the SVZ of the lateral ventricle from adult transgenic mice expressing PS1hWT or PS1ΔE9 variant. NeuN-labeled cells (red) show the architecture of LV. The insets are images of BrdU-labeled cells at a higher magnification. Scale bars: (A), 300 μm; (inset), 75 μm. B, Quantification of BrdU-labeled cells form SVZ or dentate gyrus of adult PS1hWT or PS1ΔE9 mice. Data represent m ± SEM and presented as percentage over the numbers observed in PS1hWT transgenic line. N = 5, *p < 0.05. C, SVZ NPCs cultured from adult transgenic mice expressing PS1hWT or PS1ΔE9 were lysed; total protein was Tris-Tricine SDS-PAGE resolved and immunoblotted with antibody that detects human PS1-NTF or PS1-C terminal fragment (CTF). D, In vitro proliferation rate of SVZ NPCs cultured from mentioned transgenic mice as measured by ELISA-based BrdU cell proliferation assay on days 1–5. N = 3, *p < 0.05. E, Representative images of NPC nuclei that retain BrdU [red (arrow), top and bottom] and are colabeled with nestin [green (arrow), bottom]. DAPI-stained nuclei that are negative for BrdU are in blue. Scale bar, 10 μm. F, Quantification of BrdU⁺ and nestin⁺ cells scored on days 3 and 8 in independent NPC cultures expressing PS1hWT or PS1ΔE9. Data were collected by sampling 30 nestin⁺ cells from two random fields for BrdU colabeling. N = 3, *p < 0.05.

Figure 4.

Multilineage commitment of NPCs derived from postnatal SVZ of PS1ΔE9 transgenic mice. A, Representative photomicrographs reveal the differentiation potential of NPC cultures into distinct βIII-tubulin (i; green)-, GFAP (i; red)-, MAP2 (ii; green)-, s100β (iii; green)-, and O4 (iv; green)-positive cell types, established from SVZ of adult PS1hWT or PS1ΔE9 transgenic mice. Scale bar, 25 μm. Nuclei were stained with DAPI (blue; i, iv) or propidium iodide (PI, red; ii, and iii). B, C, Quantification of differentiated progeny cell types. Number of animals per group = 5, data were obtained by scoring a minimum of 300 cells for each lineage marker, **p < 0.01. D, Representative images of NPC-differentiated βIII-tubulin [green (arrow), top] or MAP2 [green (arrow), bottom] lineage and colabeled with BrdU (red). Nuclei were stained with DAPI. Scale bar, 10 μm. E–G, Quantification of NPCs differentiated to βIII-tubulin (E), MAP2 (F), or GFAP (G) lineage and colabeled with BrdU, scored on days 3 and 8 in independent NPC cultures expressing PS1hWT or PS1ΔE9. Data were collected by sampling 100 BrdU⁺ cells from random fields for colabeling with lineage-specific differentiation. N = 3, *p < 0.05; **p < 0.01.

Figure 5.

Notch/CBF-1-dependent reporter activity in NPCs expressing FAD-linked PS1 mutants. Fold change in firefly luciferase activity in nontransgenic adult SVZ progenitors cotransduced with mentioned lentivirus encoding PS1hWT or FAD-linked PS1 mutant cDNAs and reporter lentivirus (Hes1-pomoter Luc, A; or 4×CBF1RE-reporter Luc, B). Reporter activity was normalized to total protein content across each sample and represented as fold activation over the level observed with promoterless luciferase reporter. N = 3 (A) or 4 (B), *p < 0.01.

Figure 6.

Notch/CBF-1 signaling in NPCs derived from postnatal SVZ of PS1ΔE9 transgenic mice. A, B, Fold change in firefly luciferase activity in SVZ NPCs derived from PS1hWT or PS1ΔE9 transgenic mice, transduced with either Wt-Hes1-LV-Luc, mut-Hes1-LV-Luc, Wt-4×CBF1RE-LV-Luc or Mut-4×CBF1RE-LV-Luc over Proless-Luc reporter lentivirus. NPC cultures were established from a minimum of 3 animals per group and pooled before the assay. N = 3, performed in duplicate; *p < 0.05; **p < 0.01. C, Fold change in firefly luciferase activity in SVZ NPCs derived from PS1hWT or PS1ΔE9 transgenic mice electroporated with either Hes5-promoter luciferase or promoterless luciferase construct. NPC cultures were established from a minimum of 3 animals per group. Firefly reporter activity was normalized to Renilla luciferase activity across each sample and represented as fold activation over the level observed with promoterless luciferase reporter. N = 3, performed in triplicate; **p < 0.01. D, Table summarizes the fold changes observed in Hes1, Hes5, Hes7, Hey1 and Hey2 gene transcripts in PS1ΔE9 adult SVZ NPCs relative to their levels detected in PS1hWT NPCs. Data represent m ± SEM of C _t values obtained from three independent quantitative real time RT-PCR experiments on template prepared from NPCs cultured from a minimum of N = 4 animals per group.

Figure 7.

Constitutive Notch activity restores self-renewal and differentiation defects in NPCs derived from postnatal SVZ of PS1ΔE9 transgenic mice. A, B, Histograms show the number of neurospheres ≥40 μm in diameter obtained after dissociating a single neurosphere (100 μm in diameter) cultured from SVZ of adult PS1hWT or PS1ΔE9 transgenic mice following transduction with lentivirus encoding either GFP or Notch1-ICD (A); or retrovirus encoding either GFP or HES1 cDNA (B). N = 8 (A) or 5 (B), *p < 0.05; **p < 0.01. C, Representative photomicrographs reveal the identity of LV-HANotch1-ICD-transduced, GFP⁺ NPCs (arrow), differentiated into βIII-tubulin, GFAP, s100β or MAP2 lineages. Overlay of the respective images are shown on the right of each row. Scale bar, 25 μm. D–G, Quantification of virus-transduced PS1hWT or PS1ΔE9 progenitors differentiated to βIII-tubulin⁺ or MAP2⁺ neuronal (D, E) or GFAP⁺ astroglial (F, G) progeny cell types. Data represent m ± SEM obtained from 6 independent experiments and the differentiated fate was scored by counting a minimum of 100 GFP⁺ cells or 100 DAPI⁺ nuclei that colabeled with neuronal or astrocyte cell fate marker. *p < 0.05; **p < 0.01.