Alzheimer Abeta peptide induces chromosome mis-segregation and aneuploidy, including trisomy 21: requirement for tau and APP

Abstract

Both sporadic and familial Alzheimer's disease (AD) patients exhibit increased chromosome aneuploidy, particularly trisomy 21, in neurons and other cells. Significantly, trisomy 21/Down syndrome patients develop early onset AD pathology. We investigated the mechanism underlying mosaic chromosome aneuploidy in AD and report that FAD mutations in the Alzheimer Amyloid Precursor Protein gene, APP, induce chromosome mis-segregation and aneuploidy in transgenic mice and in transfected cells. Furthermore, adding synthetic Abeta peptide, the pathogenic product of APP, to cultured cells causes rapid and robust chromosome mis-segregation leading to aneuploid, including trisomy 21, daughters, which is prevented by LiCl addition or Ca(2+) chelation and is replicated in tau KO cells, implicating GSK-3beta, calpain, and Tau-dependent microtubule transport in the aneugenic activity of Abeta. Furthermore, APP KO cells are resistant to the aneugenic activity of Abeta, as they have been shown previously to be resistant to Abeta-induced tau phosphorylation and cell toxicity. These results indicate that Abeta-induced microtubule dysfunction leads to aneuploid neurons and may thereby contribute to the pathogenesis of AD.

Figure 1.

Chromosome aneuploidy induced in transgenic mouse splenocytes carrying an AD mutant APP transgene (V717F). (A) Karyotype analysis of nontransgenic (NON) and transgenic APP+/− mice showed significantly higher levels of aneuploidy in the cells harboring a mutant APP transgene, but no increase in tetraploidy/polyploidy. In situ DNA FISH with a BAC plasmid containing mouse chromosome 16 (B) showed significantly higher levels of trisomy 16 (C) and monosomy 16 (D) in APP+/− splenocytes compared with cells from NON transgenic controls.

Figure 2.

Trisomy 16 induced in neurons of mice carrying a mutant APP transgene. Quantitative FISH analysis of resuspended cells from the whole brain of FAD mutant V717F mice costained with the NeuN antibody (A) revealed significantly higher levels of trisomy 16 compared with nontransgenic animals. Most of the aneuploidy was due to trisomy 16 neurons (B and C). No significant increase in tetrasomy 16 was detected (D).

Figure 3.

Chromosome aneuploidy induced in cells expressing either NL-APP K595N/M596L and V642I or V717F mutant APP. hTERT cells were transfected with expression vectors for the mutant human APP genes with the empty vectors pcDNA3 or pAG3 serving as control. The transfected cells were analyzed 48 h later. Expression of APP caused many cells to become aneuploid, as indicated by trisomy 21 and trisomy 12 (A–C). APP expression failed to induce a significant increase in tetrasomy for chromosome 16 (D).

Figure 4.

High levels of total aneuploidy induced in hTERT cells by either Aβ40 or Aβ42. In the series of seven experiments, metaphase karyotypes of peptide-treated hTERT cells were examined after 48 h exposure to Aβ and control peptides (A). Significant levels of aneuploidy were induced by 1 μM Aβ40 and Aβ42 compared with Aβ12-18-Scrambled peptide and Aβ42-1 reverse peptide (B).

Figure 5.

Specific chromosome aneuploidy, including trisomy 21 and trisomy 12 induced by exposure of hTERT cells to Aβ peptide. Quantitative FISH analysis with a dual color probe detecting both chromosome 21 (SpectrumOrange) and 12 (SpectrumGreen) revealed induction of trisomy 21 and 12 (A–C), but no significant increase of either tetrasomy 21 or 12 (data not shown). Comparing these results with the data of Figure 3 indicates that Aβ peptides exert a general disruptive effect on chromosome segregation during mitosis that includes but is not restricted to chromosome 21.

Figure 6.

Pretreating normal splenocytes for 3 min with 1 mM BAPTA, a chelator of extracellular Ca²⁺, showed a reduction in Aβ 1-42–induced trisomy 16 (A). Similarly, the coincubation with 1 μM of Aβ 1-42 for 48 h and 2.5 mM of LiCl, a GSK-3β inhibitor, for the last 7 h decreased trisomy 16 (B).

Figure 7.

Knocking out Tau replicates/replaces ability of Aβ to induce chromosome mis-segregation. Spleen cells from normal (WT) and Tau+/− and Tau−/− mice were cultured ± Aβ1-42 and Aβ1-40 for 48 h, and the resulting chromosome aneuploidy was assessed (A and B). Tau+/− and −/− cells displayed a higher-than-normal inherent level of aneuploidy, consistent with the requirement for Tau in the MT function in the mitotic spindle. Aβ's ability to induce chromosome mis-segregation was greatly attenuated (i.e., replaced) in Tau+/− and Tau−/− cells.

Figure 8.

Knocking out APP in the target cells prevents Aβ from inducing further chromosome mis-segregation and aneuploidy. Spleen cells from APP−/− mice were cultured ± Aβ1-42, Aβ1-40, and scrambled Aβ1-42 for 48 h, and the resulting chromosome 16 aneuploidy was assessed. Aβ induced aneuploidy in the normal (NON) cells (p = 0.01), but failed (p = 0.4) to increase aneuploidy over background in the APPKO cells. Interestingly, like the Tau−/− cells examined in Figure 7, APP−/− cells also exhibited higher levels of aneuploidy compared with nontransgenic cells (p < 0.05).