DNA replication precedes neuronal cell death in Alzheimer's disease

Abstract

Alzheimer's disease (AD) is a devastating dementia of late life that is correlated with a region-specific neuronal cell loss. Despite progress in uncovering many of the factors that contribute to the etiology of the disease, the cause of the nerve cell death remains unknown. One promising theory is that the neurons degenerate because they reenter a lethal cell cycle. This theory receives support from immunocytochemical evidence for the reexpression of several cell cycle-related proteins. Direct proof for DNA replication, however, has been lacking. We report here the use of fluorescent in situ hybridization to examine the chromosomal complement of interphase neuronal nuclei in the adult human brain. We demonstrate that a significant fraction of the hippocampal pyramidal and basal forebrain neurons in AD have fully or partially replicated four separate genetic loci on three different chromosomes. Cells in unaffected regions of the AD brain or in the hippocampus of nondemented age-matched controls show no such anomalies. We conclude that the AD neurons complete a nearly full S phase, but because mitosis is not initiated, the cells remain tetraploid. Quantitative analysis indicates that the genetic imbalance persists for many months before the cells die, and we propose that this imbalance is the direct cause of the neuronal loss in Alzheimer's disease.

Fig. 1.

The appearance of the hippocampus and basal nucleus in Alzheimer's disease. A, Loss of hippocampal pyramidal cells is evident in this cresyl violet-stained field from AD case 3 when compared with control (B). A similar reduction in cell density is visible in the large neurons of the basal nucleus of Meynert (C), compared with control (D). Immunocytochemistry reveals presence of various cell cycle-related proteins, including the DNA polymerase subunit, PCNA (E), and the G₂ cyclin, cyclin B (F).

Fig. 2.

Chromosomal location of the probes used in this study. The arrows indicate the approximate location of the probes displayed on chromosomal drawings copied from the NCBI web site (http://www.ncbi. nlm.nih.gov/LocusLink/).

Fig. 3.

Localization of FISH probes in cultured cells. Pictured here is a metaphase spread of colcemid-blocked human lymphocytes hybridized with the BACE1 BACs.A, There are two spots (arrows) of hybridization in most interphase cells. B, Note the location of the specific hybridization (arrows) to the end of a condensed chromosome 11 in metaphase cells. Scale bar, 10 μm.

Fig. 4.

Normal diploid chromosomal complement is found in most neurons at most ages. In each panel, arrowsindicate specific spots of hybridization. A,BACE1 BAC hybridization to cerebellar granule cells of newborn human cerebellum (propidium iodide counterstain). The high density of cells in the internal granule cell layer results in the apparent overlap of cells in these 10 μm paraffin sections.B, BACE1 hybridization to hippocampal pyramidal cells of a nondemented 40-year-old female. C, Hippocampal neuron from area CA4 of a control case hybridized with theBACE1 probe. Two or fewer spots of hybridization are seen. The bright yellow/orange fluorescent material in the cell cytoplasm is lipofuscin, which autofluoresces at the wavelengths used. Note that in each of these control situations, we find two or fewer spots of hybridization per cell. Scale bars, 10 μm.

Fig. 5.

FISH applied to neurons in affected areas of Alzheimer's disease brain. A, Confocal image of a hippocampal neuron from area CA3 of an Alzheimer's disease brain (case 9). Note the presence of four bright spots of fluorescence (arrows) in the nucleus, suggesting a doubling of the number of chromosomes 11 in this cell. B, Hippocampal neuron from area CA4 of the same case (9). Three spots of bright hybridization are found in this single nucleus, revealing aneuploidy for this portion of chromosome 11. C, Neuron from the motor cortex of the same Alzheimer's disease case (9) hybridized with the same BACE1 probe. D, Granule cells from the cerebellum of an Alzheimer's disease brain. In these neurons, as in all others that we examined from this region, two or fewer spots of hybridization (arrows) were found. A was taken on a Zeiss confocal microscope.B–D were taken at 1000× on a Leitz fluorescence microscope. Scale bars: A, D, 10 μm.

Fig. 6.

Different probes from different chromosomes all reveal aneuploidy in hippocampal neurons in Alzheimer's disease cases.A, Hippocampal pyramidal cell from area CA4 of Alzheimer's disease case 9 probed with the BACE2 probe. Thearrows indicate four spots of specific fluorescence found in the nucleus. B, A cell from the same region of the same case hybridized with the chromosome 21 centromeric probe. Three spots of hybridization are clearly visible in the nucleus.C, A CA3 hippocampal pyramidal neuron from control brain. Two spots of hybridization (arrows) were found when the same chromosome 21 centromeric probe was used. No cell with more than two spots was found in this or any other control case.D, A confocal image of a cell from Alzheimer's disease case 9 hybridized with the probe to the centromere of chromosome 18. Note the three hybridization spots in the nucleus of this cell. Scale bar, 10 μm.

Fig. 7.

Aneuploidy in the large neurons of the basal nucleus of Meynert. A, B, Neurons illustrated here were probed with either BACE1(A) or BACE2 (B) probes. In each case, examples of polyploid neurons were found in Alzheimer's disease but not control brains. C, A control brain probed with the BACE1 probe from chromosome 11. This field is typical of the results in the control cases in which two or fewer spots of FISH hybridization (arrows) were found. Scale bar, 10 μm.