Overexpression in neurons of human presenilin-1 or a presenilin-1 familial Alzheimer disease mutant does not enhance apoptosis

Abstract

Programmed cell death, or apoptosis, has been implicated in Alzheimer's disease (AD). DNA damage was assessed in primary cortical neurons infected with herpes simplex virus (HSV) vectors expressing the familial Alzheimer's disease (FAD) gene presenilin-1 (PS-1) or an FAD mutant of this gene, A246E. After infection, immunoreactivity for PS-1 was shown to be enhanced in infected cells. The infected cells exhibited no cytotoxicity, as evaluated by trypan blue exclusion and mitochondrial function assays. Quantitative analysis of cells that were immunohistochemically labeled using a Klenow DNA fragmentation assay or the TUNEL method revealed no enhancement of apoptosis in PS-1-infected cells. This result was confirmed using assays for chromatin condensation and for DNA fragmentation. Expression of PS-1 protected against induction of apoptosis in the cortical neurons by etoposide or staurosporine. The specificity of this phenotype was demonstrated by the fact that cortical cultures infected with recombinant HSV vectors expressing the amyloid precursor protein (APP-695) showed, in contrast, a significant increase in the number of apoptotic cells and an increase in DNA fragmentation for all parameters tested. Our results indicate that overexpression of wild-type or A246E mutant PS-1 does not enhance apoptosis in postmitotic cortical cells and suggest that the previously reported enhancement of apoptosis by presenilins may be dependent on cell type.

Fig. 1.

High levels of efficiency of expression are seen after recombinant HSV infection of cortical neurons. Mouse cortical neurons were maintained in Neurobasal medium with B27 supplement and 1% horse and fetal bovine serum. At 5 DIV, cells were infected with recombinant HSV viruses at a range of moi. A–C, HSV/Lac infection of primary cortical neurons at an moi of 2 (A) or 5 (B). Twelve hours after infection, cells were fixed and processed for β-galactosidase activity (blue cells). C, LacZ-positive and total cells were counted in 10 random fields for each moi, and the percentage of blue cells was determined. The data are expressed as the average ± SE of 10 microscopic fields, with a total of 553–641 cells counted for each moi. D–G, HSV/myc-PS-1 infection of primary cortical neurons at an moi of 1 (D) or 5 (E). The brown reaction product delineates myc immunoreactivity in infected cells. F, G, Uninfected neurons incubated with Myc antibody and processed in parallel with the cells shown in D and E.D–F are bright-field images. G is a phase-contrast image of F. Scale bar, 25 μm.

Fig. 2.

PS-1 and APP-695 overexpression in cortical cells. Primary neuronal cells prepared from E18 rat cortex were infected with recombinant HSV expressing APP-695, wild-type PS-1, or A246E-mutant PS-1, or were mock-infected. A, Cell extracts from cortical neurons that were infected with HSV/APP-695 (lanes 1 and 2, moi = 0.5 and 1, respectively) or HSV/Lac (lanes 3 and 4, moi = 0.5 and 1, respectively) or that were mock-infected (lanes 5 and 6) were analyzed by immunoblot analysis using 22C11 (Boehringer Mannheim). The cluster of bands around 110 kDa represent immature and mature (differentially glycosylated) APP-695. B, Cell extracts from cortical neurons that were infected with HSV/PS-1 (lanes 1 and 2, moi = 0.5 and 1, respectively), HSV/A246E-PS-1 (lanes 3 and4, moi = 0.5 and 1, respectively), or HSV/Lac (lanes 5 and 6, moi = 0.5 and 1, respectively) or that were mock-infected (lanes 7 and8) were analyzed by immunoblot analysis using the rabbit polyclonal 347 antiserum, against PS-1 amino acids 2–12. Full-length PS-1 and the N-terminal fragment (NTF) are immunodetected at 49 and 29 kDa, respectively. The band at 34 kDa probably is nonspecific. Detection of the full-length PS-1 bands was inhibited by the cognate peptide and was heat-sensitive (data not shown). At the exposure times used, endogenous full-length PS-1 was not detected.

Fig. 3.

Cortical neurons infected with HSV/PS-1 (A, D), HSV/A246E-PS-1 (B, E), or HSV/Lac (C, F) at an moi of 2 are viable. Twenty-four hours after infection, neurons were tested for exclusion of trypan blue. All HSV-infected neurons showed exclusion of trypan blue.A–C are phase-contrast images. D–F are bright-field images.

Fig. 4.

HSV-mediated expression of wild-type or mutated presenilins does not affect neuronal survival. Neurons were infected in parallel with HSV expressing wild-type PS-1, A246 PS-1, or LacZ, or vehicle only (Mock) at moi ranging from 0.1 to 5. Survival was measured by metabolic conversion of tetrazolium salt into formazan salt using a Cell Titer 96 Assay Kit. A, Twelve hours after infection; B, 24 hr after infection;C, 72 hr after infection. A representative result from one of three independent experiments is shown. Each value is the average of three different wells, with error bars representing the range.

Fig. 5.

Single-stranded DNA breaks in cells of recombinant HSV-infected cultures were assessed using a Klenow assay. The dense reaction product (arrows) identifies apoptotic nuclei. Control cells incubated without the Klenow enzyme give negative results (no staining). Cortical cultures infected for 24 hr at an moi of 2 with HSV/PS-1 (A) or HSV/A246E-PS-1 (B) show very few apoptotic nuclei. Cultures infected under the same conditions with HSV/APP-695 (C) show numerous apoptotic nuclei.D, LacZ-infected cells; E, mock-infected cells.

Fig. 6.

Quantification of the data shown in Figure 5 shows that overexpression of wild-type or A246E-mutant PS-1 does not enhance apoptosis in cortical cells, as measured with a Klenow DNA fragmentation assay. However, cells infected with HSV/APP-695 show a significant increase (p < 0.05; Bonferroni multiple t test) in percentage of apoptotic cells. Cortical cells were infected as described in the legend to Figure 5. The total number of cells and the number of apoptotic cells were counted in 10 random fields, and the number of apoptotic cells was expressed as a percentage of the total cells counted. The results are the average ± SEM. A total of 200–300 cells were counted for each moi. * denotes a significant (p < 0.05) difference in the percentages of apoptotic cells in HSV/APP-695-infected cultures versus HSV/Lac-infected cultures.A, Twelve hours after infection; B, 24 hr after infection.

Fig. 7.

Quantification of apoptosis using a DNA fragmentation enzyme-immunoassay (Cell Death Detection ELISA), which detects histone-associated DNA fragments that are generated during the internucleosomal degradation of genomic DNA that occurs in apoptosis. Neurons were infected in parallel with HSV/PS-1, HSV/A246E-PS-1, HSV/APP-695, or HSV/Lac, or vehicle only (mock) at moi ranging from 1 to 5. Samples were analyzed photometrically at 405 nm, using substrate solution as a reference blank. Only neurons infected with HSV/APP-695 show significant changes in DNA fragmentation. A, Twelve hours after infection; B, 24 hr after infection. Results are the average of three different wells; error bars represent range.

Fig. 8.

Overexpression of wild-type or A246E-mutated PS-1 does not enhance apoptosis in cortical cells, as detected with a TUNEL method (Apoptag). This method, in contrast to the Klenow fragmentation assay, detects primarily double-stranded DNA breaks. Cortical cells were infected as described in the legend to Figure 5. Cells were fixed and incubated with anti-digoxigenin antibodies coupled to peroxidase, and the brown reaction product was detected with DAB. For each experiment, nonspecific labeling was determined by omitting TdT. Few apoptotic cells were detected (arrows) 24 hr after infection with HSV expressing wild-type (A) or mutated (B) PS-1. An increased in apoptotic cells was observed in cells infected with HSV expressing APP-695 (C). Cells infected with HSV/Lac (D) or incubated with the vehicle (E, mock) show very few apoptotic cells.

Fig. 9.

Quantification of the percentages of apoptotic cells shown in Figure 8. Apoptotic cells were counted as described in the legend to Figure 7. Enhanced apoptosis was not observed after infection of the neurons with HSV/PS-1 or HSV/A246E-PS-1, but it was observed when the cells were infected with HSV/APP-695.A, Twelve hours after infection; B, 24 hr after infection. The results are the average ± SEM of counts from 10 microscopic fields with a total of 200–300 cells counted per moi. * denotes significance (p < 0.05) in the difference in values between cells infected with HSV/APP-695 and cells infected with HSV/Lac.

Fig. 10.

Bisbenzimide intercalates into DNA strands, allowing for fluorescent visualization of normal or condensed chromatin. Normal nuclei are regular in shape and round, whereas apoptotic nuclei (arrows) show condensation of chromatin material. Cortical cells infected with HSV/PS-1 (A) or HSV/A246E-PS-1 (B) for 24 hr at an moi of 2 show infrequent alterations in nuclear morphology. Cells infected with HSV/APP-695 (C) show an increase in the number of nuclei with condensed chromatin material. Cells infected with HSV/Lac (D) or incubated with vehicle alone (E) show similar nuclear morphology as those infected with HSV/PS-1 or HSV/A246E-PS-1.

Fig. 11.

Quantification of apoptotic cells with bisbenzimide shows a lack of enhanced apoptosis 24 hr after infection with HSV/PS-1 or HSV/A246E-PS-1. Enhanced apoptosis was observed 24 hr after infection with HSV/APP-695 at an moi of 2. The total number of cells and the number of nuclei with condensed chromatin were counted in 10 random fields, and each value was expressed as the percentage of nuclei with condensed chromatin relative to the number of total nuclei counted. The results are the average ± SEM. A total of 400–500 cells were counted for each moi. * denotes significance (p < 0.05) in the difference in values between cells infected with HSV/APP-695 and cells infected with HSV/Lac. A, Twelve hours after infection;B, 24 hr after infection.

Fig. 12.

Expression of PS-1 or A246E-PS-1 in cortical neurons protects against apoptosis induced with etoposide or staurosporine. Twelve hours after infection of cortical cells with HSV/PS-1, HSV/A246E-PS-1, or HSV/Lac at an moi of 2, cells were exposed to etoposide (ET, 20 μm) or staurosporine (Stau, 1 μm) for 5 hr. In an additional control, mock-infected cells were exposed to the same drugs. Samples were analyzed photometrically as described in Figure 7. Both etoposide and staurosporine induced a modest, albeit significant increase in DNA fragmentation in cortical cells (p < 0.05). This increase in DNA fragmentation was prevented if cells were infected with HSV/PS-1 or with HSV/A246E-PS-1 before being exposed to the apoptosis-inducing agents.