Aneuploidy and DNA replication in the normal human brain and Alzheimer's disease

Abstract

Reactivation of the cell cycle, including DNA replication, might play a major role in Alzheimer's disease (AD). A more than diploid DNA content in differentiated neurons might alternatively result from chromosome mis-segregation during mitosis in neuronal progenitor cells. It was our objective to distinguish between these two mechanisms for aneuploidy and to provide evidence for a functional cell cycle in AD. Using slide-based cytometry, chromogenic in situ hybridization, and PCR amplification of alu-repeats, we quantified the DNA amount of identified cortical neurons in normal human brain and AD and analyzed the link between a tetraploid DNA content and expression of the early mitotic marker cyclin B1. In the normal brain, the number of neurons with a more than diploid content amounts to approximately 10%. Less than 1% of neurons contains a tetraploid DNA content. These neurons do not express cyclin B1, most likely representing constitutional tetraploidy. This population of cyclin B1-negative tetraploid neurons, at a reduced number, is also present in AD. In addition, a population of cyclin B1-positive tetraploid neurons of approximately 2% of all neurons was observed in AD. Our results indicate that at least two different mechanisms need to be distinguished giving rise to a tetraploid DNA content in the adult brain. Constitutional aneuploidy in differentiated neurons might be more frequent than previously thought. It is, however, not elevated in AD. In addition, in AD some neurons have re-entered the cell cycle and entirely passed through a functional interphase with a complete DNA replication.

Figure 1.

Intermethod reliability of three independent methods for single-cell DNA quantification. A set of 48 identical microscopically identified neurons of the entorhinal cortex in a patient with early AD was evaluated through subsequent application of SBC, CISH, and PCR amplification of alu repeats. Tissue sections were first processed for SBC, followed by hybridization with the chromosome 17 probe. Subsequently, identified neurons were captured through laser microdissection and subjected to PCR amplification of alu repeats. A–C, Regression analyses reveal correlation coefficients according to Bravais-Pearson of r = 0.92 for the LSC data versus hybridization results (CISH; A), of r = 0.80 for LSC versus PCR amplification (B) and r = 0.78 for PCR amplification versus hybridization (C). All correlation coefficients are significant at p < 0.001. D, The combined set of data obtained with all three methods.

Figure 2.

Confocal image of a brain slice as used for SBC analysis. Immunohistochemical double staining for neurofilament (SMI 311, blue) and cyclin B1 (green) of pyramidal neurons in the human entorhinal cortex, counter reacted with PI (red). Scale bars, 200 μm.

Figure 3.

Quantification of neuronal loss and single-neuron DNA amount by SBC. A, Analysis of neuron number showing the decline with progression of the disease. B, Neuronal cyclin B1 expression is elevated in advanced AD stages. C–E, Single-cell DNA amount in neurons of the entorhinal cortex in controls and patients with AD. Shift of neurons from diploid toward triploid and tetraploid DNA amount in AD cases. Tetraploid neurons express cyclin B1 in AD, but not in control cases. F, Distribution of entorhinal cortical neurons with a tetraploid content of DNA according to the presence or absence of cyclin B1 expression. In both control groups, only cyclin B1-negative neurons were present, which are reduced in number in AD cases. In addition, a cyclin B1-positive cell population appears in AD and represents the majority of tetraploid cells under disease conditions. Statistics were performed for control versus AD groups with an additional analysis for early AD versus advanced AD in B. Shown are mean values ± SEM. **p ≤ 0.01; *p ≤ 0.05, Student's t test.

Figure 4.

CISH signals with a chromosome 17 probe in lymphocytes, HeLa cells and neurons of the entorhinal cortex in one advanced case of AD. A, Two spots are present in 99% of all lymphocytes. B, Proliferating HeLa cells show either two or four spots, indicating a diploid and tetraploid DNA amount. C–F, Neurons in an advanced case of AD showing one, two, three, or four spots (arrows). Scale bars, 10 μm.

Figure 5.

Quantitation of CISH signals in neurons of the entorhinal cortex in both control and AD groups. AD cases compared with controls show the same percentage of cells without or with one spot, but a shift of neurons from two spots toward three and four spots. Statistics were performed for control versus AD groups. Mean values ± SEM are shown. ***p ≤ 0.001; **p ≤ 0.01, Student's t test. In validation experiments, the probe was applied to a proliferating culture of HeLa cells and to PFA-fixed lymphocytes pretreated identically to brain slices.

Figure 6.

A, Frequency distribution of single-neuron DNA content determined by PCR amplification of alu repeats in both control and AD groups. Shift toward higher DNA content in AD cases compared with controls and appearance of a second peak displaying a doubled DNA content are shown (i.e., tetraploid neurons). B, PI integral histograms of single-neuron DNA content obtained by SBC. Each case is represented by a single graph. Similarly to the PCR data, a shift toward a higher DNA content is observed in AD.

Figure 7.

PCR analysis of the DNA content in layer V pyramidal neurons of the entorhinal (EC) and occipital cortices (OC) in six control and six AD cases to exclude interindividual differences in alu repeats. Contrary to controls, the average DNA content of entorhinal neurons was elevated compared with occipital neurons in AD cases. Shown are mean values ± SEM of 10–15 cells per specimen. * p ≤ 0.05, Student's t test.

Figure 8.

Synoptic overview on results of the current approach to identify cycling neurons in the adult human brain based on the simultaneous determination of single-neuron DNA content and expression of cyclin B1. Cyclin B1 expression occurs at the end of S phase and reaches peak expression during G2 and M phase. Neurons with a DNA amount of >2n that simultaneously express cyclin B1 are likely to have progressed in an active cycle beyond the S phase. In AD, ∼2% of neurons are tetraploid expressing cyclin B1, indicating complete replication, whereas ∼20% express cyclin B1 and contain DNA between 2n and 4n, indicating incomplete replication.