Free Radic Biol Med. 2013 Oct;63:115-25.

Alzheimer’s disease-associated polymorphisms in human OGG1 alter catalytic activity and sensitize cells to DNA damage.

Jacob KD, Noren Hooten N, Tadokoro T, Lohani A, Barnes J, Evans MK.

Laboratory of Epidemiology and Population Sciences, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224-6825, USA.

 

Abstract

Brain tissues from Alzheimer’s disease (AD) patients show increased levels of oxidative DNA damage and 7,8-dihydro-8-oxoguanine (8-oxoG) accumulation. In humans, the base excision repair protein 8-oxoguanine-DNA glycosylase (OGG1) is the major enzyme that recognizes and excises the mutagenic DNA base lesion 8-oxoG. Recently, two polymorphisms of OGG1, A53T and A288V, have been identified in brain tissues of AD patients, but little is known about how these polymorphisms may contribute to AD. We characterized the A53T and A288V polymorphic variants and detected a significant reduction in the catalytic activity for both proteins in vitro and in cells. Additionally, the A53T polymorphism has decreased substrate binding, whereas the A288V polymorphism has reduced AP lyase activity. Both variants have decreased binding to known OGG1 binding partners PARP-1 and XRCC1. We found that OGG1(-/-) cells expressing A53T and A288V OGG1 were significantly more sensitive to DNA damage and had significantly decreased survival. Our results provide both biochemical and cellular evidence that A53T and A288V polymorphic proteins have deficiencies in catalytic and protein-binding activities that could be related to the increase in oxidative damage to DNA found in AD brains. Published by Elsevier Inc.

PMID: 23684897

 

Supplements:

Alzheimer’s Disease (AD) is the most common age associated form of neurodegeneration that results in dementia.  Worldwide it is estimated that more than 35 million individuals are affected with AD, and this number is expected to double within the next 20 years.  A familial form of AD, that affects individuals between the ages of 30-60, represents less than 5% of the total cases and is linked to one of three inherited genes.  The remaining cases are not considered a natural part of aging and to date the genetic causes sporadic disease remain unclear.  One hypothesis to explain the mutations observed in AD is linked to the accrual of spontaneous mutations in the DNA of genes that are associated with AD.  One cause of spontaneous mutation is oxidative stress.

Oxidative stress occurs when cells are unable to detoxify themselves from or repair various reactive species or compounds, which are produced from both exogenous (environmental – pollution, UV exposure, nutrition) and endogenous (normal cellular respiration) sources.  The persistence of oxidative stress in a cell will ultimately cause oxidative damage of the DNA, resulting in what is referred to as a DNA lesion.  When cells cannot remove or repair the lesions within DNA the result can be a spontaneous mutation.  The presence of mutations in DNA can act to alter or block both DNA replication (copying of DNA) and transcription (the process of turning DNA into RNA).  Altering or blocking replication and transcription will result in altered protein biosynthesis (protein production).  When protein structure is altered many normal cellular pathways can be affected (signaling pathways, cellular metabolism, cell cycle function and regulation), leading to cellular dysfunction, senescence, and ultimately cell death.

When thinking about AD and how oxidative stress could produce mutations that result in disease pathology, there are a number of reasons why the brain is particularly susceptible to oxidative stress including: 1) the brain has the most exposure to oxygen of all organs in the body (accounting for only 2% of body weight, but for 20% of oxygen production).  This results in more opportunities for production of reactive species; 2) neuronal cell membranes contain components which are particularly vulnerable to oxidation to produce reactive species; 3) neurotransmitter metabolism (the breakdown of chemicals required to transmit electrical signals in the brain) produces reactive species; 4) neurons are post-mitotic (meaning they do not replicate or divide).  By not entering the cell cycle (where cell replication occurs) neurons are not able to repair certain types of DNA damage; 5) increased iron levels are found in brain tissues of AD patients.  When high levels of iron are present, chemical bonding can occur producing reactive species; and 6) low levels of antioxidant enzymes (molecules that can help prevent reactive species from being formed) have been detected in brain tissues.  It has also been shown that oxidative DNA damage is one of the earliest detectable events in AD pathogenesis.

The most common lesion formed as a result of oxidative stress is 7,8-dihydro-8-oxoguanine (8-oxoG), which can mispair with adenine, resulting in G to T and A to C mutations in the genome.  Various studies have shown that brain tissues from AD patients show increased levels of both nuclear and mitochondrial DNA damage and specifically, increases in 8-oxoG lesions were reported.

There are mechanisms in place to help cells combat the possibility of introducing mutations, which are referred to as the DNA repair pathways.  The various repair pathways are activated to deal with a specific type of DNA damage that cell has encountered.  In particular, the Base Excision Repair (BER) system is equipped to handle the oxidative damage that occurs, such as the 8-oxoG lesion.  BER is initiated by a DNA glycosylase, which is able to scan along the DNA and recognize the oxidative lesion.  The glycosylase then acts to remove the lesion, allowing the correct base to be added into the DNA.  In humans, the main glycosylase involved in repairing 8-oxoG lesions is 8-oxoguanine-DNA glycosylase (OGG1).

In addition to the wildtype (non-altered) OGG1 protein, alternative forms have been identified, called polymorphic proteins.  Overall, 21 alterations in the OGG1 protein have been identified, 13 of which are related to different diseases.  Two of the polymorphic forms,  A53T and A288V, of OGG1 have been specifically identified from brain tissues of AD patients.  Although an initial study showed that both of these polymorphic proteins have decreased activity (meaning they are less able to remove oxidative lesions), a more detailed mechanism has not been established.  We have chosen to characterize these polymorphic OGG1 proteins to determine the cause of their decreased activity and determine the consequence they have on cells.

Shown in Figure 1 is a schematic showing the consequence of an oxidative lesion in DNA.  The pathway on the left pictures a situation where the oxidative lesion is not repaired.  The oxidative lesion, represented by the letter “O”, remains in the DNA.  Through continued DNA replication, the oxidative lesion will mispair with adenine (A) (one of the 4 DNA bases), which will eventually pair with the correct base thymine (T).  In this case, the initial DNA sequence with a “G” has been mutated and this mutation will be propagated into all daughter cells.  The center pathway depicts the situation where BER is active.  OGG1 is able to sense the oxidative lesion, bind, and excise the “O” in the DNA.  The correct base can then be incorporated, resulting in no change to the DNA.

Michele K. Evans-pic1

By performing various biochemical assays we have determined that the decreased activity observed for the two AD related polymorphic OGG1 proteins are functionally different reasons (depicted in the pathway on the right in Figure 1).   In the presence of A53T polymorphic protein, we determined that the OGG1 protein is unable to bind to the DNA at the site of the oxidative lesion.  The result in this situation is the same as that where there is no DNA repair.  The oxidative lesion persists and results in a mutation.  When the OGG1 proteins expresses the A288V polymorphism we found that the OGG1 protein is still able to sense and bind to the DNA lesion.  However, in this case the OGG1 protein is unable to remove the oxidative lesion.  Once OGG1 is no longer bound to the DNA, the lesion persists and can result in a mutation.  Given that these polymorphisms have been identified only in AD patients, and it has been reported that oxidative damage accumulates in brains of individuals with AD, it is possible to speculate that in some individuals, the presence of these polymorphisms in OGG1 could result in accumulation of oxidative damage in brain tissue leading to AD.

We believe that the findings of this study show that the two polymorphisms in OGG1 that have been associated with AD have multiple functional defects that render cells more vulnerable to accumulation of oxidative damage.  Further studies are needed to determine if the presence of these polymorphisms in an individual can lead to AD susceptibility.

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