An ancestral non-proteolytic role for presenilin proteins in multicellular development of the social amoeba Dictyostelium discoideum

Abstract

Mutations in either of two presenilin genes can cause familial Alzheimer's disease. Presenilins have both proteolysis-dependent functions, as components of the γ-secretase complex, and proteolysis-independent functions in signalling. In this study, we investigate a conserved function of human presenilins in the development of the simple model organism Dictyostelium discoideum. We show that the block in Dictyostelium development caused by the ablation of both Dictyostelium presenilins is rescued by the expression of human presenilin 1, restoring the terminal differentiation of multiple cell types. This developmental role is independent of proteolytic activity, because the mutation of both catalytic aspartates does not affect presenilin ability to rescue development, and the ablation of nicastrin, a γ-secretase component that is crucial for proteolytic activity, does not block development. The role of presenilins during Dictyostelium development is therefore independent of their proteolytic activity. However, presenilin loss in Dictyostelium results in elevated cyclic AMP (cAMP) levels and enhanced stimulation-induced calcium release, suggesting that presenilins regulate these intracellular signalling pathways. Our data suggest that presenilin proteins perform an ancient non-proteolytic role in regulating intracellular signalling and development, and that Dictyostelium is a useful model for analysing human presenilin function.

Keywords: Dictyostelium; Presenilin; γ-secretase.

Fig. 1.

The Dictyostelium PsenA and PsenB proteins show structural similarity to human presenilins. (A) Bayesian-derived phylogeny of presenilin amino-acid-sequence data from animal and plant species and D. discoideum. Human SPPLA2A was used as the outgroup. Numbers adjacent to nodes represent Bayesian posterior probability values. Scale bar: the number of substitutions per site. (B) Schematic of both Dictyostelium presenilin proteins showing the putative orientation in the membrane, cytosolic and transmembrane regions, the conserved catalytic residues and the proline-alanine-leucine (PAL) sequence.

Fig. 2.

Dictyostelium presenilin proteins redundantly control multicellular development that is complemented by expression of human presenilin 1. Following starvation, wild-type (WT) cells undergo development over a 24-h period, leading to the formation of fruiting bodies; left, aerial view at low magnification; right, side view at higher magnification. Ablation of both presenilin genes (psenA⁻/psenB⁻) in one cell line gives rise to a block in fruiting-body formation, leading to a small round structure lacking a stalk. Overexpression of GFP-tagged Dictyostelium PsenB or human PSEN1 in these cells restores development, whereas PSEN2 expression partially rescues development. Scale bars: 1 mm.

Fig. 3.

Expression of PsenB or PSEN1 can rescue the block in multiple differentiation pathways resulting from the ablation of presenilin activity in the psenA⁻/psenB⁻ mutant. (A) Stalk-cell production is unaffected by ablation of either presenilin individually but is reduced when both presenilins are removed and is restored by expression of PsenB or PSEN1. (B) Spore production is reduced by ablation of either presenilin gene individually, is blocked in psenA⁻/psenB⁻ mutants and is restored by expression of PsenB or PSEN1. Insets show light microscopy images of the cell type analysed in each panel. (C) The expression of developmentally regulated genes (0, 6, 12 and 24 h post-starvation), using quantitative transcriptional analysis for the early developmental marker cAR1 and the cell-type-specific marker genes (pre-stalk, ecmA; pre-spore, psA) in wild-type and in psenA⁻/psenB⁻ cells, and following complementation with PsenB or PSEN1. Increased cAR1 expression and reduced ecmA expression caused by the loss of both presenilin genes is restored by the expression of PsenB or PSEN1. All data are presented as the mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001.

Fig. 4.

The Dictyostelium and human presenilins localise to the ER in Dictyostelium cells. The GFP tag on PsenB and PSEN1 proteins indicates that both proteins colocalise with the ER marker calnexin (labelled with a specific antibody). Blue, 4′,6-diamidino-2-phenylindole (DAPI) staining. Scale bar: 10 µm.

Fig. 5.

Dictyostelium fruiting-body development does not require the proteolytic activity of presenilins or a complete γ-secretase complex. (A) Expression of Dictyostelium PsenB^D348A/D394A or human PSEN1^D257A/D385A, lacking the key catalytic aspartic-acid residues that are necessary for proteolytic activity, rescued Dictyostelium development in psenA⁻/psenB⁻ cells. The ablation of nicastrin (ncstn⁻) had no effect on Dictyostelium fruiting-body (developmental) morphology. Images show low-magnification aerial view (left) and high-magnification side view (right). Scale bars: 1mm. (B) Spore production in psenA⁻/psenB⁻ cells is restored by expression of either Dictyostelium or human presenilins mutated at the two catalytic aspartic-acid residues and is unaffected by the ablation of nicastrin (ncstn⁻). WT, wild type. Results show means±s.e.m. Inset shows a light microscopy image of the cell type analysed. (C) Both human presenilins, when expressed in Dictyostelium psenA⁻/psenB⁻ cells, undergo endoproteolysis to yield a single band of 20–25 kDa, corresponding to the C-terminal fragment, as demonstrated by western analysis. Molecular-mass markers in kilodaltons are indicated on the left; -, untransformed psenA⁻/psenB⁻ control; BD6, mouse-blastocyst-like cells showing processing of endogenous mouse presenilins. The antibodies used for western blotting (WB) are indicated below the blots.

Fig. 6.

Loss of presenilin proteins in Dictyostelium alters cAMP and calcium levels. (A) cAMP levels in cells during growth (t = 0 h) and early development (t = 4 and 8 h) in wild-type (WT) cells and psenA⁻/psenB⁻ cells with and without complementation (rescue) with PsenB or PSEN1. Data are derived from five independent experiments and show a statistically significant increase in cAMP in psenA⁻/psenB⁻ and the PSEN1-rescue cells at 8 h only. (B) Schematic representation of the calcium-response recording prior to and upon 50 nM cAMP stimulation; A, the time until calcium response after cAMP stimulation; B, the length of the calcium response. The magnitude of the cAMP-induced calcium release is also shown. Data for A and B are presented in supplementary material Fig. S4. (C) The magnitude of the calcium response in wild-type and presenilin-mutant cells. The magnitude of the calcium response is significantly increased in psenA⁻, psenB⁻ and psenA⁻/psenB⁻ cells when compared to wild-type cells. Values shown are means±s.e.m, n = 5. *P<0.05, **P<0.01, ***P<0.001.

Fig. 7.

The Dictyostelium presenilins are functional in mammalian cells. The PsenA and PsenB proteins were expressed in mouse-blastocyst-derived BD8 cells that have both psen1 and psen2 ablated. γ-secretase activity was assessed by the fold increase in luminescence resulting from cleavage of the substrate ΔEN1 (a membrane-tethered Notch1 lacking the extracellular domain) by transfected human presenilin 1 (WT) or Dictyostelium presenilin proteins (PsenA and PsenB) with or without a C-terminal FLAG tag. Results show means±s.e.m. Data represent two to three independent experiments carried out in duplicate (n = 4–6). *P<0.05, **P<0.01, ***P<0.001.