Presenilin-1 mutations reduce cytoskeletal association, deregulate neurite growth, and potentiate neuronal dystrophy and tau phosphorylation

Abstract

Mutations in presenilin genes are linked to early onset familial Alzheimer's disease (FAD). Previous work in non-neuronal cells indicates that presenilin-1 (PS1) associates with cytoskeletal elements and that it facilitates Notch1 signaling. Because Notch1 participates in the control of neurite growth, cultured hippocampal neurons were used to investigate the cytoskeletal association of PS1 and its potential role during neuronal development. We found that PS1 associates with microtubules (MT) and microfilaments (MF) and that its cytoskeletal association increases dramatically during neuronal development. PS1 was detected associated with MT in the central region of neuronal growth cones and with MF in MF-rich areas extending into filopodia and lamellipodia. In differentiated neurons, PS1 mutations reduced the interaction of PS1 with cytoskeletal elements, diminished the nuclear translocation of the Notch1 intracellular domain (NICD), and promoted a marked increase in total neurite length. In developing neurons, PS1 overexpression increased the nuclear translocation of NICD and inhibited neurite growth, whereas PS1 mutations M146V, I143T, and deletion of exon 9 (D9) did not facilitate NICD nuclear translocation and had no effect on neurite growth. In cultures that were treated with amyloid beta (Abeta), PS1 mutations significantly increased neuritic dystrophy and AD-like changes in tau such as hyperphosphorylation, release from MT, and increased tau protein levels. We conclude that PS1 participates in the regulation of neurite growth and stabilization in both developing and differentiated neurons. In the Alzheimer's brain PS1 mutations may promote neuritic dystrophy and tangle formation by interfering with Notch1 signaling and enhancing pathological changes in tau.

Fig. 1.

PS1 associates with MT in neuronal growth cones. Confocal images show the distribution of PS1 (A) and Bip (B) in 3 DIV hippocampal growth cones. PS1 colocalizes with Bip at the central region of the growth cone. Note that PS1 IF extends to filopodia and lamellipodia (A). Cultures were stained sequentially with PSN2 and anti-Bip antibodies (see Materials and Methods). After TX-100 extraction, PS1 IF remains in the cytoskeletal preparation (C), whereas Bip IF is abolished completely (D). Shown is double IF for PS1 with antibody 2025 (E) and β-tubulin class III (F). PS1 IF appears closely associated with the microtubular network. Partial colocalization between PS1 and individual MT can be observed (arrows, E, F). A mild nocodazole treatment (see Materials and Methods) abolished PS1 (G) and β-tubulin class III (H) IF from neuronal growth cones of TX-100-extracted cultures. Note the presence of PS1 IF in the neuritic shaft where intact MT are still present (G, H). The perimeter of the growth cone before nocodazole treatment is shown in H. Scale bar, 10 μm.

Fig. 2.

PS1 associates with MF in growth cone filopodia and lamellipodia. Saponin extraction preserves the localization of PS1 (labeled with antibody AD3L) in lamellipodia (A), in which numerous MF are stained by phalloidin conjugated with Texas Red (B). Note several points of contact between PS1 and MF (arrows, A–C). Depolymerization of MF with cytochalasin B abolished PS1 (D) and phalloidin (E) IF from the periphery of saponin-extracted growth cones, whereas PS1 IF remained in the central region of the growth cones (D). Scale bar, 10 μm.

Fig. 3.

The cytoskeletal association of PS1 increases during neuronal development, and PS1 mutations reduce cytoskeletal binding. A, Western blot analysis of cytoskeletal preparations of 1, 6, and 10 DIV cultures. Shown are insoluble (Insol) and soluble (Sol) fractions prepared from triplicate cultures. All lanes were loaded with 10 μg of protein. The blots were developed with antibody AD3L that recognizes PS1 FL and CTF. Note the dramatic increase in the association of FL and CTF to the insoluble fraction during neuronal development. In contrast, the ER-resident protein Bip is solubilized completely by extraction with TX-100 (B).C, Cytoskeletal binding of RFP and PS1 expressed as the amount of protein in the insoluble-over-soluble fractions, determined by quantitative Western blot analysis with antibody AD3L to PS1 CTF and anti-RFP (see Materials and Methods). Cultures were transfected at 8 DIV and analyzed at 10 DIV. Similar results were obtained with antibodies PSN2 and 2025 recognizing PS1 NTF (data not shown). Note that PS1-wt is retained more efficiently in the cytoskeleton than PS1 mutations. Values are the mean ± SE; n = 6 independent experiments. *p < 0.001 relative to PS1-wt by Student's t test.

Fig. 4.

PS1 enhances nuclear translocation of NICD and reduces neurite growth, whereas PS1 mutations inhibit both effects.A, Confocal images of double IF labeling (merged images) showing the subcellular localization of endogenous Notch1 (green) and β-tubulin class III (red) in hippocampal neurons at 3 and 10 DIV. Diffuse Notch1 IF is detected in cell bodies and in the initial portion of neuritic processes but is excluded from neuronal nuclei (arrow) at 3 DIV; however, clear Notch1 nuclear staining is observed in the neurons by 10 DIV (arrow). Scale bar, 20 μm. B, Relative intensity of Notch1 IF in nuclear-over-cytoplasmic compartment in 3 and 10 DIV neurons transfected with the indicated plasmids at 1 and 8 DIV, respectively. IF ratios in individual cells were determined as described in Materials and Methods. At 3 DIV the PS1-wt-transfected cells showed a significant increase in nuclear IF as compared with RFP- or PS1 mutant-transfected cells. At 10 DIV the neurons expressing PS1 mutations exhibited significantly lower levels of nuclear IF than RFP- or PS1-wt-expressing cells. Values are the mean ± SE; n = 80 cells analyzed per condition in a representative experiment. Similar results were obtained in five independent experiments. *p< 0.01 relative to control RFP by Student's t test.C, Total neuritic length of 3 and 10 DIV neurons transfected with the indicated plasmids at 1 and 8 DIV, respectively. PS1 mutations failed to reduce neuritic growth in 3 DIV cultures, whereas at 10 DIV the neurons expressing PS1 mutations exhibited significantly longer processes than RFP- or PS1-wt-transfected cells. Morphometric analysis was performed as described in Materials and Methods. Values are the mean ± SE; n = 110 cells analyzed per condition in a representative experiment. Similar results were obtained in five independent experiments. *p < 0.01 relative to control RFP by Student'st test.

Fig. 5.

PS1 mutations potentiate neuronal dystrophy in cultures treated with fibrillar Aβ. A, Quantification of the number of dystrophic neurons in transfected cultures treated with Aβ. At 8 DIV the cells were treated with 20 μmfibrillar Aβ 2 hr after transfection and were analyzed at 10 DIV (see Materials and Methods). In nontransfected cultures (No Transf.) the number of dystrophic neurons is expressed as a percentage of the total number of neurons in the culture. In transfected cultures the number of transfected dystrophic neurons is expressed as a percentage of the total number of transfected cells. Note the significant increase of dystrophic neurons expressing PS1 mutations as compared with RFP-transfected and PS1-wt-transfected ones. Cultures treated with reverse Aβ peptide or vehicle alone did not develop neuritic dystrophy. Values are the mean ± SE;n = 80 cells scored per condition in a representative experiment. Similar results were obtained in five independent experiments. *p < 0.01 relative to control RFP by Student's t test. B, Morphology of 10 DIV hippocampal neurons expressing PS1 mutations treated with reverse sequence Aβ peptide (Control) or Aβ. Note the aberrant morphology developed by Aβ-treated neurons. Scale bar, 20 μm.

Fig. 6.

PS1 mutations increase the number of dystrophic neurites per cell in Aβ-treated cultures. A, Confocal images of neurons transfected with the indicated PS1 mutations and treated with Aβ. Red IF represents RFP autofluorescence, and the blue is Aβ IF detected with antibody 6E10. The images show examples of abnormal neuritic branches protruding from neuronal cell bodies in contact with Aβ deposits (arrows). Scale bar, 20 μm. B, Serial confocal sections (I, II, III) of cells transfected with PS1-I143T and PS1-D9 showing dystrophic processes in contact with Aβ. Dystrophic neurites grow upward, entering inside Aβ deposits and extending several micrometers above the initial focal plane. Scale bar, 5 μm. C, Quantification of the number of dystrophic events per neuron induced by 20 μmAβ in 10 DIV cells transfected with the indicated constructs. PS1 mutations markedly increased the number of dystrophic processes per neuron. Morphometric analysis was performed as described in Materials and Methods. Values are the mean ± SE; n = 30 individual cells scored per condition in a representative experiment. Similar results were obtained in four independent experiments. *p < 0.01 relative to control RFP by Student'st test.

Fig. 7.

PS1 colocalizes and coimmunoprecipitates with tau, and PS1 mutations increase Aβ-induced tau phosphorylation.A, Double IF showing colocalization of endogenous PS1 (PSN2) and tau (Tau-1) in neuronal growth cones. Note the presence of tau IF in filopodia (arrows). Scale bar, 10 μm.B, Cell lysates of 10 DIV hippocampal neurons treated with Aβ were immunoprecipitated with PHF-1 (IPP PHF-1), blotted, and stained with antibodies 2025 and mAD3L. Antibody 2025 recognized PS1 NTF in PHF-1-immunoprecipitated material. Antibody 2025 also labeled PS1 FL and NTF in a homogenate of transfected Cos cells (Cos PS1wt) used as a positive control. Antibody mAD3L labeled PS1 FL and CTF in a homogenate of transfected Cos cells and recognized a band corresponding to PS1 FL in PHF-1-immunoprecipitated material. Cell lysate immunoprecipitated with nonimmune mouse sera (IPP non-imm) showed no reaction with 2025 and mAD3L antibodies. Preabsorption of both 2025 and mAD3L with the corresponding antigenic peptides completely abolished PS1-specific labeling in the Western blot (Preab). Secondary antibodies used to develop the immunoblots reacted with PHF-1 IgG in the immunoprecipitated material (IgG).C, Cell lysates of 10 DIV hippocampal neurons treated with Aβ were immunoprecipitated with anti-PS1 antibody 2025, blotted, and reacted with PHF-1, which recognizes phosphorylated tau at Ser 396/404. PHF-1 labeled a 55 kDa band in a homogenate of Aβ-treated neurons (Aβ) and a band of similar molecular weight in the material immunoprecipitated with 2025 (IPP-PS1 2025). Cell lysate immunoprecipitated with nonimmune rabbit sera (IPP non-imm) showed no reaction with PHF-1.D, Western blot analysis of transfected cultures treated with Aβ. Cultures transfected at 8 DIV were treated with 20 μm Aβ and harvested at 10 DIV. Protein (10 μg) was loaded in each lane. The blots were developed with PHF-1 and Tau-1 antibodies. Expression of green fluorescent protein (GFP) was used as a control. Note the increase in PHF-1 staining in cultures expressing PS1 mutations. No significant changes in the levels of nonphosphorylated tau were detected by Tau-1. E, Relative changes in the level of phosphorylated tau in Aβ-treated cultures expressing PS1 mutations detected by the PHF-1 antibody. Quantitative Western blot analysis was performed as described in Materials and Methods. Values are the mean ± SE; n = 5 independent experiments. *p < 0.01 relative to PS1-wt by Student's t test.