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DNA Damage and Senescence in the Aging and Alzheimer's Disease Cortex Are Not Uniformly Distributed - PubMed

  • ️Mon Jan 01 2024

DNA Damage and Senescence in the Aging and Alzheimer's Disease Cortex Are Not Uniformly Distributed

Gnanesh Gutta et al. Biomedicines. 2024.

Abstract

Alzheimer's disease (AD) is a neurodegenerative illness with a typical age of onset exceeding 65 years of age. The age dependency of the condition led us to track the appearance of DNA damage in the frontal cortex of individuals who died with a diagnosis of AD. The focus on DNA damage was motivated by evidence that increasing levels of irreparable DNA damage are a major driver of the aging process. The connection between aging and the loss of genomic integrity is compelling because DNA damage has also been identified as a possible cause of cellular senescence. The number of senescent cells has been reported to increase with age, and their senescence-associated secreted products are likely contributing factors to age-related illnesses. We tracked DNA damage with 53BP1 and cellular senescence with p16 immunostaining of human post-mortem brain samples. We found that DNA damage was significantly increased in the BA9 region of the AD cortex compared with the same region in unaffected controls (UCs). In the AD but not UC cases, the density of cells with DNA damage increased with distance from the pia mater up to approximately layer V and then decreased in deeper areas. This pattern of DNA damage was overlaid with the pattern of cellular senescence, which also increased with cortical depth. On a cell-by-cell basis, we found that the intensities of the two markers were tightly linked in the AD but not the UC brain. To test whether DNA damage was a causal factor in the emergence of the senescence program, we used etoposide treatment to damage the DNA of cultured mouse primary neurons. While DNA damage increased after treatment, after 24 h, no change in the expression of senescence-associated markers was observed. Our work suggests that DNA damage and cellular senescence are both increased in the AD brain and increasingly coupled. We propose that in vivo, the relationship between the two age-related processes is more complex than previously thought.

Keywords: aging; p16; prefrontal cortex; γ-H2AX.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1

Representative images of amyloid deposit density in AD and UC brains. (A) Radial fields are shown spanning the entire distance from the pia mater (top) to the white matter (bottom) in the sampled regions of BA9. The left panel illustrates the 6E10 immunostaining (green) pattern in UC material (Braak 0–II). The deposits of amyloid are small and relatively sparse. The right panel illustrates a comparable image of an AD case. Note the increased density of 6E10-positive deposits found largely in the upper third of the cortex. (B) A higher magnification of a similar section from a UC case immunostained with the indicated antibodies. (C) A comparable field from an AD case showing the dense nature of the amyloid deposits labeled with 6E10 (red). MAP2 (green) and DAPI (blue) were labeled as well. Scale bar = 50 µm.

Figure 2
Figure 2

Typical images of the middle layers of the BA9 cortex immunostained for two DNA damage markers, 53BP1 (red) and γ-H2AX (green), plus MAP2 (white). Note the near total overlap in the nuclei of immunostained cells. (A) Low-magnification view of a region from an unaffected control (UC). (Ai) The same field showing only γ-H2AX and MAP2 staining. (Aii) The same field showing only 53BP1 and MAP2 staining. (B,Bi,Bii) Higher magnification of the areas indicated by the blue boxes. (C,Ci,Cii) Images comparable to those shown in (A), but showing a region from an Alzheimer’s disease brain. (D,Di,Dii) Images comparable to those shown in (B), but showing a region from an Alzheimer’s disease brain. Scale bar = 50 µm.

Figure 3
Figure 3

Examples of human brain cryostat sections immunostained for DNA damage (53BP1—red) and senescence (p16—green) with a DAPI (blue) nuclear counterstain. The panels on the left (A,C) are from an individual who died with no cognitive impairment. The panels on the right (B,D) are from a person who died with advanced Alzheimer’s dementia. Different cortical depths are illustrated for both cases. The top panels (A,B) are from a more superficial location (Zone 2); the bottom panels (C,D) are from a location closer to the white matter (Zone 5). The insets in each figure (e.g., Ai and Aii) are higher magnifications of the cells indicated by the white box. Scale bar = 20 µm. The white arrows indicate examples of cells with a diffuse nuclear staining pattern of p16; the yellow arrows indicate cells with a clumped cytoplasmic p16 pattern.

Figure 4
Figure 4

(A) A low-magnification image of a MAP2-immunostained section from a human UC (Case 5794) BA9 (location indicated in the inset diagram). The red lines illustrate the six cortical zones. The leftmost zone that is closest to the pia is defined as Zone 1 and the rightmost zone adjacent to the white matter is defined as Zone 6. (B) A high-magnification image of the area indicated by the white box in panel (A) immunostained for 53BP1. (C) The field of cells shown in panel (B) overlaid with the detections identified by QuPath. Inner circles represent the location of the DAPI-stained nuclei; outer circles represent the “pseudoplasm” calculated as a 5 µm extension of the nuclear outline. Red detections represent cells identified as 53BP1-positive; grey detections represent 53BP1-negative cells. (D) A high-magnification image of Zone 5 in an age-matched human AD case stained with 53BP1. (E) The field of cells shown in panel (D) overlaid with the detections identified by the same QuPath parameters as in panel (C). (F) Average density of DAPI+ nuclei in all AD or UC cases. (G) Average density of 53BP1+ nuclei in all AD or UC cases. Error bars in (F,G) = SEM. AD: n = 4 cases. UC: n = 3 cases. (ns = not significant; ** p < 0.01).

Figure 5
Figure 5

(A) Average density of 53BP1+ cells in the six cortical zones of the UC (blue symbols) and AD (red symbols) cases. Error bars = SEM. AD: n = 4 cases. UC: n = 3 cases. (B) UC data from (A) replotted to show the least-squares fit relationship (thick line, r2 = 0.124) and the quadratic fit (thin line, r2 = 0.155; adjusted r2 = 0.043) with 95% confidence limits. The least-squares method gave a slightly better fit to the data, but the slope was not significantly different from zero (p = 0.1522). (C) AD data from (A) replotted to show the least-squares fit relationship (thin line, r2 = 0.40) and the quadratic fit (thick line, r2 = 0.71) with 95% confidence limits. The quadratic equation gave a much better fit to the data. (D) A representative plot of 53BP1 intensity as a function of nuclear size. The X-axis represents the ranking of over 12,000 cells from Zone 4 by the area of their nuclei (from smallest to largest). Each point represents the average of 100 cells. The Y-axis is the average intensity of 53BP1 staining in successive groups of 100. Blue lines = unaffected control cases. Red lines = Alzheimer’s disease cases. * = p < 0.05, *** = p < 0.001.

Figure 6
Figure 6

(A) Average intensity of p16 immunostaining in the nuclei of cells identified as 53BP1+ in both UC (blue) and AD (red) cases. ** p < 0.01. (B) Average intensity of nuclear p16 immunostaining of 53BP1+ cells in the six cortical zones of the UC (blue symbols; r2 = 0.2429 with significant non-zero slope (p = 0.0377)) and AD (red symbols; r2 = 0.7024 with significant non-zero slope (p < 0.0001)) cases. (C) Average intensity of “pseudoplasmic” p16 immunostaining of 53BP1+ cells in the six cortical zones of the UC (blue symbols; r2 = 0.1958 with nonsignificant non-zero slope (p > 0.05)) and AD (red symbols; r2 = 0.4843 with significant non-zero slope (p = 0.0002)) cases. (D) Average intensity of nuclear p16 immunostaining of 53BP1+-negative cells in the six cortical zones of the UC (green symbols) and AD (purple symbols) cases. Error bars in (A–D) = SEM. AD: n = 4 cases. UC: n = 3 cases. (E) A plot of nuclear p16 immunostaining intensity as a function of 53BP1 immunostaining intensity from a random sampling of 100 cells/case from UC (blue symbols; n = 3) and AD (red symbols; n = 4) cases. The equations for the least-squares fit of the two datasets are indicated. (F) Immunohistochemistry showing the relationship and colocalization of p16 and 53BP1 in the nuclei. Green arrows indicate cells positive for p16 only. Red arrows indicate cells positive for 53BP1 only. Yellow arrows indicate cells positive for both p16 and 53BP1.

Figure 7
Figure 7

(A) Representative fields of DIV14 cultures of mouse embryonic cortical neurons immunostained for MAP2 (cyan), p27 (red), and γ-H2AX (green) and counterstained with DAPI (blue). The top row of images is from a control culture (n = 3); the middle row is from cultures treated for 24 h with 1 µM of etoposide (n = 3); and the bottom row is from cultures treated with 10 µM of etoposide (n = 3). Left column = merge; middle column = γ-H2AX plus DAPI; right column = p27 plus DAPI. (B) The fraction of γ-H2AX-positive cells in control and etoposide-treated cultures (** p < 0.01; *** p < 0.001). (C) The fraction of p27-positive cells in control and etoposide-treated cultures. Error bars in (B,C) = SEM. (D) A plot of p27 immunostaining intensity as a function of γ-H2AX immunostaining intensity of cells in untreated cultures (n = 3). (E) p27 immunostaining intensity as a function of γ-H2AX immunostaining intensity of cells in cultures treated with 10 µM of etoposide for 24 h (n = 3).

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