Aging is the strongest risk factor for Alzheimer’s disease, but exactly what it is about aging that underlies the pathology of AD, or cognitive decline in general, is a question that still confounds researchers. One theory, for example, suggests that neurons, which are terminally differentiated and so don’t undergo cycles of DNA replication and proofreading, are slouches when it comes to DNA repair and accumulate a disproportionate burden of genetic mutations over time (see ARF recent live discussion ). Now, in the June 9 online version of Nature, Bruce Yankner and colleagues at Children’s and Brigham and Women’s hospitals, both in Boston, provide experimental evidence to back up this hypothesis. Going further, they offer a new conceptual framework that links DNA damage in gene promoters to altered expression of genes in the aging brain. They also articulate a new concept of “vulnerable genes” that contain sequences which predispose to DNA damage. Some of these vulnerable genes are known to play critical roles in learning and memory.
First author Tao Lu and colleagues examined postmortem frontal cortex samples taken from normal donors aged 26 to 106 years. Lu analyzed these samples, 30 in all, with Affymetrix gene arrays to measure how the expression of approximately 11,000 genes changed with age. Compared with people younger than 42, Lu found that in people older than 76, about 4 percent of genes are differentially expressed in the frontal cortex—some up- and some downregulated. The latter included genes involved in synaptic plasticity (e.g., NMDA and AMPA receptor subunits), long-term potentiation (e.g., calmodulin CaM kinase II and protein kinase C), vesicular transport (e.g., synaptobrevin, dynein, and Rab GTPases), and microtubular function (MAP1B, MAP2, and tau), while upregulated genes included those that mediate stress responses, such as chaperones and antioxidant enzymes. DNA repair genes, such as the 8-oxoguanine DNA glycosylase, which excises damaged nucleotides, were also upregulated. The paper includes a table of differentially expressed genes representating functional categories; a complete list is provided in the Supplementary Information.
The induction of DNA repair mechanisms led Yankner and colleagues to wonder if this pattern of gene expression may be tied to DNA damage. To test this, Lu et al. devised an assay that could resolve DNA damage in specific gene sequences. They isolated genomic DNA under conditions that prevent in-vitro oxidation and incubated it with enzymes that specifically excise damaged bases, particularly 8-oxoguanine. Then, using the real-time polymerase chain reaction, they were able to identify and quantify stretches of damaged DNA by their failure to amplify (excision of the damaged base creates a single strand break that inhibits PCR).
Lu and collegues then applied this approach to brain samples spanning the entire adult age range and found that DNA damage was present in every gene examined in aging brain. The twist is that damage was greatest in promoter regions. These regions may be hardest hit because they are not subject to transcription-coupled repair, the major repair mechanism in mature neurons. (Promoter regions are usually repaired by a process that requires transit through the cell cycle.) Of 30 promoters examined, many showed an age-related increase in DNA damage by age 40, and all genes showed damage by 70. The clincher came when the researchers compared promoter damage with age-related changes in gene expression: They found that those genes that were downregulated had significantly more damage than genes that were upregulated or unchanged. The results suggest an association between promoter DNA damage and age-related changes in gene expression, the authors note.
To determine whether there is a causal relationship between damage and gene expression, Lu and colleagues examined DNA damage in cultured human neuroblastoma cells and primary human cortical neurons treated with a mild oxidative stress (hydrogen peroxide and ferric chloride) that did not kill the cells. Here again, genes that were downregulated in aging brains e.g. the tau and calmodulin 1 genes, were damaged and transcriptionally repressed to a greater extent than genes that did not change in the aged brain.(e.g. GAPDH and beta tubulin). To validate this correlation between vulnerable genes in vitro and in vivo, the authors examined gene promoters in human cortical neurons subjected to oxidative stress. Transfection of the DNA repair enzyme reversed both the damage and transcriptional downregulation .
Why do some promoters appear more vulnerable than others? To approach this question, Lu et al. cloned the promoters from brain genomic DNA into luciferase reporter plasmids and damaged them in vitro by treatment with hydrogen peroxide or irradiation with ultraviolet light. The promoters that show increased damage and reduced transcription in the aged brain also showed increased damage and reduced transcription in vitro. Furthermore, when these promoter constructs were transfected into neuronal cells they showed reduced base excision DNA repair. These experiments suggest that the vulnerability of these genes is a function of their DNA sequence, as opposed to some difference in signaling pathways or cellular responses to stress.
Other experiments in the paper examined the reduction in expression of some mitochondrial genes that the authors observed in the aging brain. They mimicked these gene expression changes in cultured neuronal cells using siRNA, and found that this increased DNA damage to vulnerable nuclear genes. These findings suggest that one source of DNA-damaging free radicals in the aging brain might be dysfunctional mitochondria.
The “findings suggest that accelerated DNA damage may contribute to reduced gene expression in the human brain after age 40,” and that “genome damage may compromise systems that subserve synaptic function and neuronal survival” write the authors. The also authors suggest that this could be a starting point for trying to understand why aging of the brain is the major risk factor for AD —Tom Fagan
Q&A with Bruce Yankner
Q: How does this view of aging fit in with the amyloid hypothesis of AD?
A: The amyloid hypothesis does not provide a clear explanation for why age is the major risk factor for Alzheimer's disease. This also belies our basic ignorance about the initial events that underlie sporadic AD. This gap in our understanding of AD reflects, in my view, a major gap in our understanding of the molecular basis of aging in the human brain.
Q: Do you think that the very nature of the samples, i.e., postmortem, may factor into the reduced expressions and DNA damage you observed?
A: We also examined expression and DNA damage in intracortical biopsy samples from elective neurosurgical procedures. Although the number of these samples was limited, the results were quite similar to those obtained for age-matched postmortem samples. To directly assess the role of postmortem interval, we performed linear regression analysis among all the postmortem intervals and expression changes in the two age-related gene clusters overall, or for 20 individual age-downregulated and 20 individual age-upregulated genes. Neither of these analyses showed a statistically significant relationship between postmortem interval and expression level. This may reflect the fact that we did not use tissue from brains with long postmortem intervals. For the DNA damage assays, it was important to isolate DNA using conditions that prevent in-vitro oxidation, e.g., including a free-radical scavenger and purging buffers with nitrogen.
Q: Are similar changes occurring in non-brain tissues?
A: We plan to examine blood cells and skin fibroblasts, but do not yet have results. We have examined other brain regions, specifically hippocampus, and find similarities that suggest to us that there may be a global program of brain aging that is superimposed on region-specific changes.
Q: Will it ever be possible to repair age-related damage to DNA?
A: We found that DNA repair enzymes can restore the expression of vulnerable genes damaged in cell culture, raising the whimsical possibility that some aspects of brain aging may be reversible.
Q: What about the genes that are upregulated with aging? Could DNA damage explain this, too?
A: Some genes are upregulated in the aging brain with a time course that parallels the time course of DNA damage. One possible mechanism could involve inactivation of motifs for transcriptional repressors. Alternatively, DNA damage could affect histone modification and interfere with gene silencing, although we don’t have evidence for this.
Q: What about AD and other diseases. Will you look at tissue samples to estimate how DNA damage may correlate with neurodegeneration?
A: Experiments along those lines are ongoing.
Q: Have you any theories as to why some promoters are affected and not others?
A: This is a fundamental mechanistic question. Our preliminary studies raise the possibility that specific guanine-rich motifs may be “hot spots” of oxidative DNA damage. This may be a function of the specific sequence as well as the GC content. For example, the beta tubulin promoter, which is relatively resistant to damage, and the CaM1 promoter, which is more sensitive, have similar overall guanine content, but the spatial distribution of GC-rich sequences relative to the transcription start site differs.